METHOD FOR SCREENING FOR DIET PROVIDING PRODUCTION OF MILK HAVING IMMUNOREGULATORY ACTION

TECHNICAL FIELD

The present invention relates to a method for screening for a diet providing production of milk having immunoregulatory action, and use thereof. They are useful in the fields of food, feed, and so forth.

BACKGROUND ART

Exosomes are microvesicles secreted by living body cells or cultured cells, and it is known that miRNAs (microRNAs) and proteins exist therein. As body fluids in which presence of exosomes is confirmed, there are known blood (serum and plasma), breast milk, bronchoalveolar lavage fluid, malignant ascite fluid, synovial fluid, urine, amniotic fluid, sperm, saliva, and so forth. Among these body fluids, only blood serum has been investigated about RNAs so far, and all of the target RNAs are miRNAs. On the other hand, in the researches using cells other than body fluid such as cultured cells, RNAs in microvesicles derived from mast cells and glioblastoma have been investigated, and mRNAs are also investigated in these researches (Non-patent document 1 for all the technical background described in this paragraph).

As for milk, it has been reported that miRNAs are contained in bovine milk, bovine serum, and commercial powdered infant formula (Non-patent document 2), and it has been reported that miRNAs are contained in breast milk and various body fluids other than breast milk (Non-patent document 3). Further, it has been reported that miRNAs in breast milk are involved in immunoregulation (Non-patent document 4).

Furthermore, it has also been reported that miRNAs and mRNAs are contained in microvesicles derived from bovine milk (Non-patent document 5). However, mRNAs investigated in this report are only those coding for 6 kinds of proteins, α-S1-casein, α-S2-casein, β-casein, κ-casein, β-lactoglobulin, and MFG-E8, which are milk proteins, and EF-1α, which is a transcription factor.

miRNAs and mRNAs derived from exosomes in human saliva have recently been investigated, and 509 kinds of mRNAs have been detected by using a microarray (Non-patent document 6). Further, potentiality as a biomarker of renal diseases has been suggested for nucleic acids derived from exosomes in human urine, and these nucleic acids include mRNAs (Non-patent document 7).

Breast milk contains secretory IgA, lactoferrin, lysozyme, cytokines, and so forth, and it is considered that it protects infants from infection, and promotes development of infant's immunity (Non-patent document 8). Actually, it is known that children grown up on breast milk involve a lower risk of infection in the bronchi or intestinal tract compared with children not grown in such a manner. Breast milk contains IgA, lactoferrin, glycoproteins, glycolipids etc., which show antibacterial activities, as well as cytokines, which regulate immunocytes.

As mentioned above, it has been reported that miRNAs involved in immunoregulation exist in milk, and that mRNAs coding for milk proteins and transcription factor are contained in microvesicles derived from bovine milk, but it is not known that mRNAs involved in immunoregulation exist in milk.

PRIOR ART REFERENCES

Non-Patent Documents

• Non-patent document 1: Simpson R. J., Expert Rev. Proteomics, 6:267-283, 2009
• Non-patent Document 2: Chen, X. et al., Cell Res., 20:1128-1137, 2010
• Non-patent Document 3: Weber, J. A. et al., Clin. Chem., 56:1733-1741, 2010
• Non-patent document 4: Kosaka, N. et al., Silence, 1:7, 2010
• Non-patent document 5: Hata, T. et al., Biochem. Biophys. Res. Commun., 396:528-533, 2010
• Non-patent document 6: Palanisamy, V. et al., PLoS One, 3(9):e3148, 2008
• Non-patent document 7: Miranda. K. C. et al., Kidney Int., 78:191-199, 2010
• Non-patent document 8: Goldman, A. S., Breastfeed. Med., 2(4):195-204, 2007

SUMMARY OF THE INVENTION

Object to be Achieved by the Invention

An object of the present invention is to provide a method for screening for a diet providing production of milk having an immunoregulatory action, a food having an immunoregulatory action, and a method for producing it.

Means for Achieving the Object

The inventors of the present invention conducted researches with paying attention to the fact that breast milk affected maturation of infant's immune system. As a result, they found that mRNAs of immunity-related genes were contained in breast milk, and accomplished the present invention.

The present invention thus provides a method for screening for a diet or a substance providing production of breast milk having an immunoregulatory action, which comprises identifying a diet or a substance that increases or decreases amount of mRNA present in milk of a mammal by using correlation of mRNA profile in the milk and a diet ingested by the mammal or a substance contained in the diet as an index.

In an embodiment of the aforementioned method, the immunoregulatory action is an immunostimulating action, and when either one or both of the following conditions are satisfied, it is determined that the diet or the substance provides production of breast milk having an immunostimulating action:

(A) the mRNA is a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet increases amount of the mRNA,

(B) the mRNA is a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet decreases amount of the mRNA.

In another embodiment of the aforementioned method, the immunoregulatory action is an immunosuppressive action, and when either one or both of the following conditions are satisfied, it is determined that the diet or the substance provides production of breast milk having an immunosuppressive action:

(a) the mRNA is a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet decreases amount of the mRNA,

(b) the mRNA is a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet increases amount of the mRNA.

In an embodiment of the aforementioned method, mRNA profiles in the milk observed before and after ingestion of the diet are compared, and when amount of at least one kind of mRNA in the milk observed after the ingestion is higher than that observed before the ingestion, it is determined that the diet increases the amount of the mRNA in the milk.

In a preferred embodiment of the aforementioned method, mRNA profiles in the milk observed before and after ingestion of the diet are compared, and when amount of a mRNA in the milk observed after the ingestion is 1.5 times or more of that observed before the ingestion, it is determined that the diet increases the amount of the mRNA in the milk.

In another embodiment of the aforementioned method, mRNA profiles in the milk observed before and after ingestion of the diet are compared, and when amount of at least one kind of mRNA in the milk observed after the ingestion is lower than that observed before the ingestion, it is determined that the diet decreases the amount of the mRNA in the milk.

In a preferred embodiment of the aforementioned method, mRNA profiles in the milk observed before and after ingestion of the diet are compared, and when amount of a mRNA observed after the ingestion is 0.67 times or less of that observed before the ingestion, it is determined that the diet decreases the amount of the mRNA in the milk.

Further, in a preferred embodiment of the aforementioned method, the mammal is a human, a rat, or a bovine.

In a preferred embodiment of the aforementioned method, the mRNA profiles comprises amount of mRNA selected from the group consisting of Cyp24a1, Dnm1, Dpysl2, Fam89a, Arhgap22, Fbxl5, LOC304396, Btg2, Csnk1g1, Mll1, Eml4, Nelf, Aldh1l1, Boc, Usp43_predicted, Rassf5, Man2a1, Srgn, Gbf1, RGD1562551, Lrba, P2ry2, Bcl211, Rbm14, RGD1307365, Tecpr1, Kctd9, Pkig, RGD1308139, Klhl35, Gab1, Arid5b, Zfp157, Mafk, Trim47, Ddx21, Tiparp, Hspa1b, Tcfcp2l1, Nr3c2, Klf4, Dhx8, Myh14, Mcam, Dusp3, Acer2, L3 mbtl3, Fcho2, RT1-N3, Ash1l, Paxip1, Irgq, Plcb1, Sts, Spetex-2D, Zmym3, Anxa1, Rtn4ip1, Gngt1, Srcap, Otud4, RGD1307569, Adpgk, Plk3, Phf1, St14, N4bp1, Naaa, Expi, Fgf7, RGD1562492, Lama2, Ahnak, Sec3l1, Mks1, Prodh, Baz1b, R3hdm1, Setd5, Rhebl1, Kdm5b, Fbxw8, Epb4.1l4a, Lpin2, Cxcl2, Nr2c2, Ckap4, Ankrd1, Ugcg, Fcgr2b, Sf3a1, Parg, Heatr6, Ctdspl, Tp53bp1, Fam60a, Pik3r2, RGD1305089, Arhgef5, Nek7, Rpt, Vhl, Usp31, Camsap1, RGD1308759, Ldlrap1, RGD1359108, Sp1, Mast4, Rela, Nhlrc3, Intu, Gcap14, Spetex-2B, Cdc42se2, Chd7, Rbms1, Invs, LOC100294508, Kif2a, Exoc6, Zbtb1, Rnd3, Rb1cc1, Sphk1, Ugt1a2, Noxo1, Mllt6, Thumpd2, Ptpn4, Thex1, S100a8, Grk5, Trove2, Pcdh17, Med13, LOC684871, RGD1305350, Fam82a2, Rab43, Clip1, Ankrd57, RGD1563375, Crim1, Celsr2, Idh1, Lrrc61, Aff4, Arhgap1, Itpr3, Lhfp, Dnajc12, Garnl1, Gtf3c5, Hspb8, Chac1, Pof1b, Ptpre, Arl6ip1, Son, Ppargc1b, Med1, Casp12, Zbtb39, Urb1, Wdr89, Josd1, Pank4, Ska2, Rrm1, Klf11, Phc1, Birc6, Cask, Mesdc2, St5, Smyd5, Heca, Plagl2, Strn4, Ftsjd1, Maml1, RGD1561817, Anxa8, Nup153, Anxa2, Trappc2, Gpr155, Zfhx3, Morc2, Lrsam1, Polg, RGD1311307, Kcnk4, Tmcc3, RGD1562136, Pde4b, Lkap, Chn1, Dusp16, Pex5, Ns5atp9, Map6, Angptl4, Dnmt3b, Fert2, Gzf1, Plcb4, Ap4 m1, Stat3, Rkhd1, LOC691223, Dcp2, Rtn4, Tlr3, Tubb6, Casp8, Nin, Fyn, Tmem77, Maml3, Slc15a4, Sqrdl, Ppp1r9b, Pafah1b1, Def8, Fam164a, Znf746, Bmyc, RGD1305235, Thada, Hip1, Pgrmc2, Sgk3, Baz1a, Foxn3, Map3k12, Efhd2, Pogz, Pcf11, RGD1560523, Mcart1, Sox4, Calcoco1, Myom1, Mtmr9, Akap13, Rhobtb1, Matr3, LOC684297, Rxrb, Mars2, Abcb8, Bik, Med22, Cryab, Cpt1a, Cnksr1, Kirrel3, Arl13a, Sept8, Rb1, Prmt2, Ccnb3, Zhx2, Pds5b, Hdac6, Prdm4, Smarcad1, Usp9x, Sdc1, Arhgap28, Rad17, Sorbs1, Tp63, Wdr48, Hnrpll, LOC687994, Sptbn1, Dtx31, Cnnm2, Fbln5, Usp48, Tgm2, RGD1564776, RGD1563106, Taok1, Snx30, Utp6, Cytip, Rest, Eif4g3, Adnp2, Wdr1, Hiat1, Ddx58, RGD1359600, Arhgap12, Dapk2, Nup160, Eea1, LOC363188, RGD1563437, Paqr5, LOC500684, Ddhd1, Kdm6b, Ube2d1, Rhoc, LOC361100, Cx3cl1, LOC100302372, RGD1311892, Fbxo42, Daam1, K1h15, Fbxo30, Syt12, Herc1, Dab2ip, Tacc2, Sipa1l1, Ets2, Zbtb43, Fhdc1, Pdlim5, Stxbp5, Hdac8, Sestd1, Acot2, Fam117b, Nrip1, Aldh3a2, Exosc8, Shmt1, Lrrc57, Rell2, Zfyve16, Cebpd, Tnrc6b, Nsun6, Dennd5a, Fbxw5, Slc25a25, Dtwd1, Hspb1, Homer2, Nhej1, Etnk1, Nol9, Med23, Nup133, Rnd1, Rplp1, Ankfy1, Pik3r4, Plcd1, Cxcl12, Lix1l, Ralbp1, Cald1, LOC302495, Cdcp1, Ralb, Pcyt1a, Skil, Trim25, Pigv, Mapk3, Pard6g, Klf7, Pxn, Dnaja4, Cnot1, Capn2, Birc3, Frmd8, Chd2, Prpf39, Taf5, Eif4enif1, Pkn2, Fam179b, Kdm2a, Gda, Ccdc21, Il17re, Eif4e3, Dock8, Nolc1, Sult1a1, Ankrd17, Trim2, Nfib, Cd14, S100a10, LOC687105, Ccdc123, RGD1309537, Pols, Aplf, Gulp1, Slc40a1, Tfdp2, Dyrk2, Anxa5, Nr3c1, RGD1306862, Rkhd2, Cdc42 bpb, Numa1, Mocos, Mafb, Akap11, Diaph1, Znrf1, Sos1, Rhob, Pkp2, Srf, RGD1310159, Cdh1, Rnf19a, Pdlim1, Csnk1g3, Hectd1, Taf5l, Noc3l, Nfat5, RGD1562529, Atl3, Ptpn9, Nedd4l, Wipi1, Gnal, Tle1, Pprc1, Src, Clic1, Secisbp21, Rnf103, Zfp772, Ogt, Zcchc2, Clasp1, Gtpbp2, Ift172, LOC500625, RGD1309350, Map3k6, Plce1, Nedd4, Cpne3, Prim2, Trafd1, Myef2, Znf532, Tmem50b, Il6ra, Pdcd6ip, LOC500757, Tpm4, Rnasen, Lpin1, Flot1, Cds1, Tnfaip1, Mudeng, Cdc215, Arid2, Znf654, Nat8b, Snx12, Ap1g1, Itsn2, Fam21c, RGD1306820, Mesdc1, Tmco6, Fkbp5, Prpf8, Pdcd11, Runx1, Epn2, Arid4b, Slk, Prkx, Dnajb3, Phf17, Fam193a, Pvrl2, Tep1, Atxn7, Wwtr1, LOC313672, Mus81, C1d, Arid4a, Ehd1, Usp32, Carhsp1, Acss1, Ctcf, Bag3, Mtrr, Ywhah, Csrp1, Smc3, Sorbs2, RGD1309762, Ptpn1, Setd2, Efr3a, Tnk1, Ncor1, Coq4, Klhl24, Cox11, Tfip11, Rcan1, Nagk, Ep300, Myst3, Rbm28, Anapc7, Ezh1, Cyp27a1, Irs2, Sdccag1, Foxp1, Dusp18, Hcca2, Fbxl8, Chd1, Tp53 bp2, RGD1309995, RGD1310552, Kif5b, Pcaf, Slc25a42, Ocln, RGD1559961, Kifap3, Wtip, Gapvd1, Ankrd42, Supt4h1, Haus1, RGD1310951, Prdm2, Arvcf, Gtf2h1, Rassf6, Pcif1, Arap2, Ube2k, Twf1, Rpl8, Prdx1, Phrf1, Pcm1, Mapk12, Slc1a4, Prl3d4, Trim16, LOC689842, Cacna1e, RT1-EC2, Gal, Arnt2, Stk11ip, Sdc3, RGD1563155, Tp53i11, Mpz, Dnmt31, Ache, Cln5, Olr184, Asb7, Rtp3, Peli2, Caskin2, Cass4, Plvap, LOC680711, Nat2, RGD1565498, Hist1h1a, Col22a1, LOC100360528, Htr6, Trim42, Ccdc89, Atp10d, Klri2, LOC100360801, RGD1560019, Ugt2b37, Oprk1, Cyct, Pdzd2, Vom2r31, Sh2d5, Tmem136, Gh1, Ttl, Pgbd5, Zfp583, Ankrd2, Avil, Mcm3ap, Mypn, Frmd4a, Rnf182, Kcng1, Paqr7, RGD1304929, RGD1565100, Pkp1, Nnmt, Csrnp1, Siae, Hoxb6, Pga5, Hoxd9, RGD1308541, Cyth3, Fbp1, Bnc1, Col18a1, Traf3, Sema7a, Rcn3, Ift122, Olr1275, LOC619574, Slc5a4a, Smc2, Cldn18, LOC679462, Pogk, Olr445, Abhd15, LOC688219, RGD1564846, Pias4, Alg8, Nkx2-4, Mmp2, Iqcf1, B9d2, Fam107a, Rarres1, RGD1561276, Wbp2, Wdr93, C1qtnf5, Olr1303, Egfl7, Olr507, Slc32a1, RGD1563516, Id3, LOC691130, Ccdc58, Sox21, Tnfsf12, Qrich2, RGD1308930, LOC307727, B3galnt2, Tob2, Creb3l1, K1k12, Dqx1, Ncoa2, Slfnl1, Elf2, Gsx2, Hhip, Grm2, Fam170a, Pnma3, Gpc2, Npvf, RGD1308143, Tsen54, Igfbp1, Zdhhc19, Csrnp2, Dcst2, Krt1, Nanos1, Cdh13, Ebi3, Prr3, Rnf43, Btbd17, Olr1626, Trim26, Gata3, Cdkn1c, Hrc, Grasp, Galm, RGD1308106, Samd14, Olr471, Oprd1, Pim1, Il2rb, Ptpn13, Mtus1, RGD1305298, Fhl3, Olr515, Tpsab1, RGD1564961, Hdhd2, Farp1, Ptprs, Hand1, Rpl37a, Tcf15, Neurod2, Usp20, Foxo3, Aldh1l2, RGD1308706, Nov, RGD1566130, Slc34a2, Gzma, Hist1h4b, Iqgap3, Enpp2, Tm7sf2, Exo1, Slc9a1, Rpl37, RGD1565970, Pde12, Eefsec, Dmrta2, LOC498685, Brunol5, Ocm, Olr4, Tmem164, RGD1561843, Flad1, Frmd4b, Marcksl1, Bhlhe22, Yif1b, Ttc38, Phyh, Mfsd6, LOC257642, Fam189b, Sned1, LOC100362849, Cd3d, Slc25a29, Cd7, RGD1563281, Grb7, Ccdc147, Stat2, LOC688228, Ppp3ca, LOC688241, Shisa4, Atp8b1, Krtap1-5, Cd3g, Txnl4b, Ranbp10, Dynlt3, Atp2b2, Trub1, Zhx3, Rexo4, Snapc2, LOC685031, Rad54l, Vps37c, Hmga1, Olr1572, Furin, RT1-CE5, Rab15, Folr1, Fundc1, Pak3, Aqp2, Hapln2, Tmem37, Gjd3, Lamb1, Cox5a, Cfd, Jak3, Magt1, Sc4mol, Uros, Calca, Abca7, Pm20d2, Dpf2, Tmprss4, Znf467, Btbd9, Rasgrf1, RGD1310270, Adam15, Kif15, Plekho2, Atp2c2, Rps25, Lipa, Cpeb2, Znf575, LOC287010, Arhgap26, LOC311352, Znrf4, Msh5, Tmed7, Gpd1l, Ankrd37, Cox4i2, Msl3l2, Snrnp70, Rps6ka5, Tomm20, Rassf4, Hes1, Ctdsp1, Pomc, Inpp4b, Nfix, Ydjc, Cst3, Tesk2, Tcn2, RGD1309847, Artn, Wibg, Prpf4, LOC685179, Igsf3, Got1, Lcp2, Agrn, Cited2, Bat4, RGD1308059, Sft2d1, Ubac2, Scly, Wrb, Zfp161, Fads2, Fbxo8, RGD1563148, Vegfb, Lpl, Chaf1a, Srrd, Steap1, Fkbp11, Slc39a3, Zfp53, Bcan, Foxc2, Jph1, Ccl27, Pdia3, Gabrq, Surf6, Runx3, Abcb6, Cdk2, LOC687813, and Porcn.

Further, in a preferred embodiment of the aforementioned method, the mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat is selected from the group consisting of Cyp24a1, Dnm1, Dpysl2, Fam89a, Arhgap22, Fbxl5, LOC304396, Btg2, Csnk1g1, Mll1, Eml4, Nelf, Aldh1l1, Boc, Usp43_predicted, Rassf5, Man2a1, Srgn, Gbf1, RGD1562551, Lrba, P2ry2, Bcl2l1, Rbm14, RGD1307365, Tecpr1, Kctd9, Pkig, RGD1308139, Klhl35, Gab1, Arid5b, Zfp157, Mafk, Trim47, Ddx21, Tiparp, Hspa1b, Tcfcp2l1, Nr3c2, Klf4, Dhx8, Myh14, Mcam, Dusp3, Acer2, L3mbtl3, Fcho2, RT1-N3, Ash1l, Paxip1, Irgq, Plcb1, Sts, Spetex-2D, Zmym3, Anxa1, Rtn4ip1, Gngt1, Srcap, Otud4, RGD1307569, Adpgk, Plk3, Phf1, St14, N4 bp1, Naaa, Expi, Fgf7, RGD1562492, Lama2, Ahnak, Sec3l1, Mks1, Prodh, Baz1b, R3hdm1, Setd5, Rhebl1, Kdm5b, Fbxw8, Epb4.1l4a, Lpin2, Cxcl2, Nr2c2, Ckap4, Ankrd1, Ugcg, Fcgr2b, Sf3a1, Parg, Heatr6, Ctdsp1, Tp53bp1, Fam60a, Pik3r2, RGD1305089, Arhgef5, Nek7, Rp2, Vhl, Usp31, Camsap1, RGD1308759, Ldlrap1, RGD1359108, Sp1, Mast4, Rela, Nhlrc3, Intu, Gcap14, Spetex-2B, Cdc42se2, Chd7, Rbms1, Invs, LOC100294508, Kif2a, Exoc6, Zbtb1, Rnd3, Rb1cc1, Sphk1, Ugt1a2, Noxo1, Mllt6, Thumpd2, Ptpn4, Thex1, S100a8, Grk5, Trove2, Pcdh17, Med13, LOC684871, RGD1305350, Fam82a2, Rab43, Clip1, Ankrd57, RGD1563375, Crim1, Celsr2, Idh1, Lrrc61, Aff4, Arhgap1, Itpr3, Lhfp, Dnajc12, Garnl1, Gtf3c5, Hspb8, Chac1, Pof1b, Ptpre, Arl6ip1, Son, Ppargc1b, Med1, Casp12, Zbtb39, Urb1, Wdr89, Josd1, Pank4, Ska2, Rrm1, Phc1, Birc6, Cask, Mesdc2, St5, Smyd5, Heca, Plagl2, Strn4, Ftsjd1, Maml1, RGD1561817, Anxa8, Nup153, Anxa2, Trappc2, Gpr155, Zfhx3, Morc2, Lrsam1, Polg, RGD1311307, Kcnk4, Tmcc3, RGD1562136, Pde4b, Lkap, Chn1, Dusp16, Pex5, Ns5atp9, Map6, Angptl4, Dnmt3b, Fert2, Gzf1, Plcb4, Ap4m1, Stat3, Rkhd1, LOC691223, Dcp2, Rtn4, Tlr3, Tubb6, Casp8, Nin, Fyn, Tmem77, Maml3, Slc15a4, Sqrdl, Ppp1r9b, Pafah1b1, Def8, Fam164a, Znf746, Bmyc, RGD1305235, Thada, Hip1, Pgrmc2, Sgk3, Baz1a, Foxn3, Map3k12, Efhd2, Pogz, Pcf11, RGD1560523, Mcart1, Sox4, Calcoco1, Myom1, Mtmr9, Akap13, Rhobtb1, Matr3, LOC684297, Rxrb, Mars2, Abcb8, Bik, Med22, Cryab, Cpt1a, Cnksr1, Kirrel3, Arl13a, Sept8, Rb1, Prmt2, Ccnb3, Zhx2, Pds5b, Hdac6, Prdm4, Smarcad1, Usp9x, Sdc1, Arhgap28, Rad17, Sorbs1, Tp63, Wdr48, Hnrpll, LOC687994, Sptbn1, Dtx31, Cnnm2, Fbln5, Usp48, Tgm2, RGD1564776, RGD1563106, Taok1, Snx30, Utp6, Cytip, Rest, Eif4g3, Adnp2, Wdr1, Hiat1, Ddx58, RGD1359600, Arhgap12, Dapk2, Nup160, Eea1, LOC363188, RGD1563437, Paqr5, LOC500684, Ddhd1, Kdm6b, Ube2d1, Rhoc, LOC361100, Cx3cl1, LOC100302372, RGD1311892, Fbxo42, Daam1, Klhl5, Fbxo30, Syt12, Herc1, Dab2ip, Tacc2, Sipa1l1, Ets2, Zbtb43, Fhdc1, Pdlim5, Stxbp5, Hdac8, Sestd1, Acot2, Fam117b, Nrip1, Aldh3a2, Exosc8, Shmt1, Lrrc57, Rell2, Zfyve16, Cebpd, Tnrc6b, Nsun6, Dennd5a, Fbxw5, Slc25a25, Dtwd1, Hspb1, Homer2, Nhej1, Etnk1, Nol9, Med23, Nup133, Rnd1, Rplp1, Ankfy1, Pik3r4, Plcd1, Cxcl12, Lix1l, Ralbp1, Cald1, LOC302495, Cdcp1, Ralb, Pcyt1a, Skil, Trim25, Pigv, Mapk3, Pard6g, Klf7, Pxn, Dnaja4, Cnot1, Capn2, Birc3, Frmd8, Chd2, Prpf39, Taf5, Eif4enif1, Pkn2, Fam179b, Kdm2a, Gda, Ccdc21, Il17re, Eif4e3, Dock8, Nolc1, Sult1a1, Ankrd17, Trim2, Nfib, Cd14, S100a10, LOC687105, Ccdc123, RGD1309537, Pols, Aplf, Gulp1, Slc40a1, Tfdp2, Dyrk2, Anxa5, Nr3c1, RGD1306862, Rkhd2, Cdc42bpb, Numa1, Mocos, Mafb, Akap11, Diaph1, Znrf1, Sos1, Rhob, Pkp2, Srf, RGD1310159, Cdh1, Rnf19a, Pdlim1, Csnk1g3, Hectd1, Taf5l, Noc3l, Nfat5, RGD1562529, Atl3, Ptpn9, Nedd4l, Wipi1, Gnal, Tle1, Pprc1, Src, Clic1, Secisbp21, Rnf103, Zfp772, Ogt, Zcchc2, Clasp1, Gtpbp2, Ift172, LOC500625, RGD1309350, Map3k6, Plce1, Nedd4, Cpne3, Prim2, Trafd1, Myef2, Znf532, Tmem50b, Il6ra, Pdcd6ip, LOC500757, Tpm4, Rnasen, Lpin1, Flot1, Cds1, Tnfaip1, Mudeng, Cdc215, Arid2, Znf654, Nat8b, Snx12, Ap1g1, Itsn2, Fam21c, RGD1306820, Mesdc1, Tmco6, Fkbp5, Prpf8, Pdcd11, Runx1, Epn2, Arid4b, Slk, Prkx, Dnajb3, Phf17, Fam193a, Pvrl2, Tep1, Atxn7, Wwtr1, LOC313672, Mus81, C1d, Arid4a, Ehd1, Usp32, Carhsp1, Acss1, Ctcf, Bag3, Mtrr, Ywhah, Csrp1, Smc3, Sorbs2, RGD1309762, Ptpn1, Setd2, Efr3a, Tnk1, Ncor1, Coq4, Klhl24, Cox11, Tfip11, Rcan1, Nagk, Ep300, Myst3, Rbm28, Anapc7, Ezh1, Cyp27a1, Irs2, Sdccag1, Foxp1, Dusp18, Hcca2, Fbx18, Chd1, Tp53 bp2, RGD1309995, RGD1310552, Kif5b, Pcaf, Slc25a42, Ocln, RGD1559961, Kifap3, Wtip, Gapvd1, Ankrd42, Supt4h1, Haus1, RGD1310951, Prdm2, Arvcf, Gtf2h1, Rassf6, Pcif1, Arap2, Ube2k, Twf1, Rpl8, Prdx1, Phrf1, Pcm1, and Mapk12

The aforementioned mRNAs are mRNAs corresponding to the genes mentioned in Tables 1 to 14 mentioned later. In the tables, the gene names mentioned in the columns of “Gene Symbol” and “Entrez Gene ID” are the gene names used in the Entrez Gene database of NCBI (National Center for Biotechnology Information), and the gene names mentioned in the column of “Gene Name” are the generic gene names. The same shall apply to Tables 15 to 22 mentioned later.

Further, in a preferred embodiment of the aforementioned method, the mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat is selected from the group consisting of Slc1a4, Prl3d4, Trim16, LOC689842, Cacna1e, RT1-EC2, Gal, Arnt2, Stk11ip, Sdc3, RGD1563155, Tp53i11, Mpz, Dnmt31, Ache, Cln5, Olr184, Asb7, Rtp3, Peli2, Caskin2, Cass4, Plvap, LOC680711, Nat2, RGD1565498, Hist1h1a, Col22a1, LOC100360528, Htr6, Trim42, Ccdc89, Atp10d, Klri2, LOC100360801, RGD1560019, Ugt2b37, Oprk1, Cyct, Pdzd2, Vom2r31, Sh2d5, Tmem136, Gh1, Tt1, Pgbd5, Zfp583, Ankrd2, Avil, Mcm3ap, Mypn, Frmd4a, Rnf182, Kcng1, Paqr7, RGD1304929, RGD1565100, Pkp1, Nnmt, Csrnp1, Siae, Hoxb6, Pga5, Hoxd9, RGD1308541, Cyth3, Fbp1, Bnc1, Col18a1, Traf3, Sema7a, Rcn3, Ift122, Olr1275, LOC619574, Slc5a4a, Smc2, Cldn18, LOC679462, Pogk, Olr445, Abhd15, LOC688219, RGD1564846, Pias4, Alg8, Nkx2-4, Mmp2, Iqcf1, B9d2, Fam107a, Rarres1, RGD1561276, Wbp2, Wdr93, C1qtnf5, Olr1303, Egfl7, Olr507, Slc32a1, RGD1563516, Id3, LOC691130, Ccdc58, Sox21, Tnfsf12, Qrich2, RGD1308930, LOC307727, B3galnt2, Tob2, Creb3l1, K1k12, Dqx1, Ncoa2, Slfnl1, Elf2, Gsx2, Hhip, Grm2, Fam170a, Pnma3, Gpc2, Npvf, RGD1308143, Tsen54, Igfbp1, Zdhhc19, Csrnp2, Dcst2, Krt1, Nanos1, Cdh13, Ebi3, Prr3, Rnf43, Btbd17, Olr1626, Trim26, Gata3, Cdkn1c, Hrc, Grasp, Galm, RGD1308106, Samd14, Olr471, Oprd1, Pim1, Il2rb, Ptpn13, Mtus1, RGD1305298, Fh13, Olr515, Tpsab1, RGD1564961, Hdhd2, Farp1, Ptprs, Hand1, Rp137a, Tcf15, Neurod2, Usp20, Foxo3, Aldh1l2, RGD1308706, Nov, RGD1566130, Slc34a2, Gzma, Hist1h4b, Iqgap3, Enpp2, Tm7sf2, Exo1, Slc9a1, Rp137, RGD1565970, Pde12, Eefsec, Dmrta2, LOC498685, Brunol5, Ocm, Olr4, Tmem164, RGD1561843, Flad1, Frmd4b, Marcksl1, Bhlhe22, Yif1b, Ttc38, Phyh, Mfsd6, LOC257642, Fam189b, Sned1, LOC100362849, Cd3d, Slc25a29, Cd7, RGD1563281, Grb7, Ccdc147, Stat2, LOC688228, Ppp3ca, LOC688241, Shisa4, Atp8b1, Krtap1-5, Cd3g, Txnl4b, Ranbp10, Dynlt3, Atp2b2, Trub1, Zhx3, Rexo4, Snapc2, LOC685031, Rad54l, Vps37c, Hmga1, Olr1572, Furin, RT1-CE5, Rab15, Folr1, Fundc1, Pak3, Aqp2, Hapln2, Tmem37, Gjd3, Lamb1, Cox5a, Cfd, Jak3, Magt1, Sc4mol, Uros, Calca, Abca7, Pm20d2, Dpf2, Tmprss4, Znf467, Btbd9, Rasgrf1, RGD1310270, Adam15, Kif15, Plekho2, Atp2c2, Rps25, Lipa, Cpeb2, Znf575, LOC287010, Arhgap26, LOC311352, Znrf4, Msh5, Tmed7, Gpd1l, Ankrd37, Cox4i2, Msl3l2, Snrnp70, Rps6ka5, Tomm20, Rassf4, Hes1, Ctdsp1, Pomc, Inpp4b, Nfix, Ydjc, Cst3, Tesk2, Tcn2, RGD1309847, Artn, Wibg, Prpf4, LOC685179, Igsf3, Got1, Lcp2, Agrn, Cited2, Bat4, RGD1308059, Sft2d1, Ubac2, Scly, Wrb, Zfp161, Fads2, Fbxo8, RGD1563148, Vegfb, Lpl, Chaf1a, Srrd, Steap1, Fkbp11, Slc39a3, Zfp53, Bcan, Foxc2, Jph1, Cc127, Pdia3, Gabrq, Surf6, Runx3, Abcb6, Cdk2, LOC687813, and Porcn.

The aforementioned mRNAs are mRNAs corresponding to the genes mentioned in Tables 15 to 22 mentioned later.

The present invention also provides a method for producing milk or a dairy product having an immunoregulatory action, which comprises the step of giving a diet or a substance identified to increase or decrease amount of mRNA in milk of a mammal by the aforementioned screening method to a mammal (except for human), and the step of collecting milk of the mammal.

In an embodiment of the aforementioned method, the immunoregulatory action is an immunostimulating action, and the diet or the substance is one determined to provide production of breast milk having an immunostimulating action.

In another embodiment of the aforementioned method, the immunoregulatory action is an immunosuppressive action, and the diet or the substance is one determined to provide production of breast milk having an immunosuppressive action.

The present invention also provides a method for producing a composition for oral ingestion having an immunostimulating action, which comprises adding a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat to a base for a composition for oral ingestion.

In a preferred embodiment of the aforementioned method, the mRNA is selected from the group consisting of Cyp24a1, Dnm1, Dpysl2, Fam89a, Arhgap22, Fbxl5, LOC304396, Btg2, Csnk1g1, Mll1, Eml4, Nelf, Aldh1l1, Boc, Usp43_predicted, Rassf5, Man2a1, Srgn, Gbf1, RGD1562551, Lrba, P2ry2, Bcl2l1, Rbm14, RGD1307365, Tecpr1, Kctd9, Pkig, RGD1308139, Klhl35, Gab1, Arid5b, Zfp157, Mafk, Trim47, Ddx21, Tiparp, Hspa1b, Tcfcp2l1, Nr3c2, Klf4, Dhx8, Myh14, Mcam, Dusp3, Acer2, L3 mbtl3, Fcho2, RT1-N3, Ash1l, Paxip1, Irgq, Plcb1, Sts, Spetex-2D, Zmym3, Anxa1, Rtn4ip1, Gngt1, Srcap, Otud4, RGD1307569, Adpgk, Plk3, Phf1, St14, N4bp1, Naaa, Expi, Fgf7, RGD1562492, Lama2, Ahnak, Sec311, Mks1, Prodh, Baz1b, R3hdm1, Setd5, Rhebl1, Kdm5b, Fbxw8, Epb4.1l4a, Lpin2, Cxcl2, Nr2c2, Ckap4, Ankrd1, Ugcg, Fcgr2b, Sf3a1, Parg, Heatr6, Ctdsp1, Tp53bp1, Fam60a, Pik3r2, RGD1305089, Arhgef5, Nek7, Rp2, Vhl, Usp31, Camsap1, RGD1308759, Ldlrap1, RGD1359108, Sp1, Mast4, Rela, Nhlrc3, Intu, Gcap14, Spetex-2B, Cdc42se2, Chd7, Rbms1, Invs, LOC100294508, Kif2a, Exoc6, Zbtb1, Rnd3, Rb1cc1, Sphk1, Ugt1a2, Noxo1, Mllt6, Thumpd2, Ptpn4, Thex1, S100a8, Grk5, Trove2, Pcdh17, Med13, LOC684871, RGD1305350, Fam82a2, Rab43, Clip1, Ankrd57, RGD1563375, Crim1, Celsr2, Idh1, Lrrc61, Aff4, Arhgap1, Itpr3, Lhfp, Dnajc12, Garnl1, Gtf3c5, Hspb8, Chac1, Pof1b, Ptpre, Arl6ip1, Son, Ppargc1b, Med1, Casp12, Zbtb39, Urb1, Wdr89, Josd1, Pank4, Ska2, Rrm1, Klf11, Phc1, Birc6, Cask, Mesdc2, St5, Smyd5, Heca, Plagl2, Strn4, Ftsjd1, Maml1, RGD1561817, Anxa8, Nup153, Anxa2, Trappc2, Gpr155, Zfhx3, Morc2, Lrsam1, Polg, RGD1311307, Kcnk4, Tmcc3, RGD1562136, Pde4b, Lkap, Chn1, Dusp16, Pex5, Ns5atp9, Map6, Angptl4, Dnmt3b, Fert2, Gzf1, Plcb4, Ap4m1, Stat3, Rkhd1, LOC691223, Dcp2, Rtn4, Tlr3, Tubb6, Casp8, Nin, Fyn, Tmem77, Maml3, Slc15a4, Sqrdl, Ppp1r9b, Pafah1b1, Def8, Fam164a, Znf746, Bmyc, RGD1305235, Thada, Hip1, Pgrmc2, Sgk3, Baz1a, Foxn3, Map3k12, Efhd2, Pogz, Pcf11, RGD1560523, Mcart1, Sox4, Calcoco1, Myom1, Mtmr9, Akap13, Rhobtb1, Matr3, LOC684297, Rxrb, Mars2, Abcb8, Bik, Med22, Cryab, Cpt1a, Cnksr1, Kirrel3, Arl13a, Sept8, Rb1, Prmt2, Ccnb3, Zhx2, Pds5b, Hdac6, Prdm4, Smarcad1, Usp9x, Sdc1, Arhgap28, Rad17, Sorbs1, Tp63, Wdr48, Hnrpll, LOC687994, Sptbn1, Dtx31, Cnnm2, Fbln5, Usp48, Tgm2, RGD1564776, RGD1563106, Taok1, Snx30, Utp6, Cytip, Rest, Eif4g3, Adnp2, Wdr1, Hiat1, Ddx58, RGD1359600, Arhgap12, Dapk2, Nup160, Eea1, LOC363188, RGD1563437, Paqr5, LOC500684, Ddhd1, Kdm6b, Ube2d1, Rhoc, LOC361100, Cx3c11, LOC100302372, RGD1311892, Fbxo42, Daam1, Klhl5, Fbxo30, Syt12, Herc1, Dab2ip, Tacc2, Sipa1l1, Ets2, Zbtb43, Fhdc1, Pdlim5, Stxbp5, Hdac8, Sestd1, Acot2, Fam117b, Nrip1, Aldh3a2, Exosc8, Shmt1, Lrrc57, Rell2, Zfyve16, Cebpd, Tnrc6b, Nsun6, Dennd5a, Fbxw5, Slc25a25, Dtwd1, Hspb1, Homer2, Nhej1, Etnk1, Nol9, Med23, Nup133, Rnd1, Rplp1, Ankfy1, Pik3r4, Plcd1, Cxcl12, Lix1l, Ralbp1, Cald1, LOC302495, Cdcp1, Ralb, Pcyt1a, Skil, Trim25, Pigv, Mapk3, Pard6g, Klf7, Pxn, Dnaja4, Cnot1, Capn2, Birc3, Frmd8, Chd2, Prpf39, Taf5, Eif4enif1, Pkn2, Fam179b, Kdm2a, Gda, Ccdc21, Il17re, Eif4e3, Dock8, Nolc1, Sult1a1, Ankrd17, Trim2, Nfib, Cd14, S100a10, LOC687105, Ccdc123, RGD1309537, Pols, Aplf, Gulp1, Slc40a1, Tfdp2, Dyrk2, Anxa5, Nr3c1, RGD1306862, Rkhd2, Cdc42bpb, Numa1, Mocos, Mafb, Akap11, Diaph1, Znrf1, Sos1, Rhob, Pkp2, Srf, RGD1310159, Cdh1, Rnf19a, Pdlim1, Csnk1g3, Hectd1, Taf51, Noc31, Nfat5, RGD1562529, Atl3, Ptpn9, Nedd41, Wipi1, Gnal, Tle1, Pprc1, Src, Clic1, Secisbp2l, Rnf103, Zfp772, Ogt, Zcchc2, Clasp1, Gtpbp2, Ift172, LOC500625, RGD1309350, Map3k6, Plce1, Nedd4, Cpne3, Prim2, Trafd1, Myef2, Znf532, Tmem50b, Il6ra, Pdcd6ip, LOC500757, Tpm4, Rnasen, Lpin1, Flot1, Cds1, Tnfaip1, Mudeng, Cdc2l5, Arid2, Znf654, Nat8b, Snx12, Ap1g1, Itsn2, Fam21c, RGD1306820, Mesdc1, Tmco6, Fkbp5, Prpf8, Pdcd11, Runx1, Epn2, Arid4b, Slk, Prkx, Dnajb3, Phf17, Fam193a, Pvrl2, Tep1, Atxn7, Wwtr1, LOC313672, Mus81, C1d, Arid4a, Ehd1, Usp32, Carhsp1, Acss1, Ctcf, Bag3, Mtrr, Ywhah, Csrp1, Smc3, Sorbs2, RGD1309762, Ptpn1, Setd2, Efr3a, Tnk1, Ncor1, Coq4, Klhl24, Cox11, Tfip11, Rcan1, Nagk, Ep300, Myst3, Rbm28, Anapc7, Ezh1, Cyp27a1, Irs2, Sdccag1, Foxp1, Dusp18, Hcca2, Fbxl8, Chd1, Tp53bp2, RGD1309995, RGD1310552, Kif5b, Pcaf, Slc25a42, Ocln, RGD1559961, Kifap3, Wtip, Gapvd1, Ankrd42, Supt4h1, Haus1, RGD1310951, Prdm2, Arvcf, Gtf2h1, Rassf6, Pcif1, Arap2, Ube2k, Twf1, Rp18, Prdx1, Phrf1, Pcm1, and Mapk12.

The present invention also provides a method for producing a composition for oral ingestion having an immunosuppressive action, which comprises adding a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat to a base for a composition for oral ingestion.

In a preferred embodiment of the aforementioned method, the mRNA is selected from the group consisting of Slc1a4, Prl3d4, Trim16, LOC689842, Cacna1e, RT1-EC2, Gal, Arnt2, Stk11ip, Sdc3, RGD1563155, Tp53i11, Mpz, Dnmt3l, Ache, Cln5, Olr184, Asb7, Rtp3, Peli2, Caskin2, Cass4, Plvap, LOC680711, Nat2, RGD1565498, Hist1h1a, Col22a1, LOC100360528, Htr6, Trim42, Ccdc89, Atp10d, Klri2, LOC100360801, RGD1560019, Ugt2b37, Oprk1, Cyct, Pdzd2, Vom2r31, Sh2d5, Tmem136, Gh1, Ttl, Pgbd5, Zfp583, Ankrd2, Avil, Mcm3ap, Mypn, Frmd4a, Rnf182, Kcng1, Paqr7, RGD1304929, RGD1565100, Pkp1, Nnmt, Csrnp1, Siae, Hoxb6, Pga5, Hoxd9, RGD1308541, Cyth3, Fbp1, Bnc1, Col18a1, Traf3, Sema7a, Rcn3, Ift122, Olr1275, LOC619574, Slc5a4a, Smc2, Cldn18, LOC679462, Pogk, Olr445, Abhd15, LOC688219, RGD1564846, Pias4, Alg8, Nkx2-4, Mmp2, Iqcf1, B9d2, Fam107a, Rarres1, RGD1561276, Wbp2, Wdr93, C1qtnf5, Olr1303, Egfl7, Olr507, Slc32a1, RGD1563516, Id3, LOC691130, Ccdc58, Sox21, Tnfsf12, Qrich2, RGD1308930, LOC307727, B3galnt2, Tob2, Creb3l1, Klk12, Dqx1, Ncoa2, Slfnl1, Elf2, Gsx2, Hhip, Grm2, Fam170a, Pnma3, Gpc2, Npvf, RGD1308143, Tsen54, Igfbp1, Zdhhc19, Csrnp2, Dcst2, Krt1, Nanos1, Cdh13, Ebi3, Prr3, Rnf43, Btbd17, Olr1626, Trim26, Gata3, Cdkn1c, Hrc, Grasp, Galm, RGD1308106, Samd14, Olr471, Oprd1, Pim1, Il2rb, Ptpn13, Mtus1, RGD1305298, Fhl3, Olr515, Tpsab1, RGD1564961, Hdhd2, Farp1, Ptprs, Hand1, Rp137a, Tcf15, Neurod2, Usp20, Foxo3, Aldh1l2, RGD1308706, Nov, RGD1566130, Slc34a2, Gzma, Hist1h4b, Iqgap3, Enpp2, Tm7sf2, Exo1, Slc9a1, Rp137, RGD1565970, Pde12, Eefsec, Dmrta2, LOC498685, Brunol5, Ocm, Olr4, Tmem164, RGD1561843, Flad1, Frmd4b, Marcksl1, Bhlhe22, Yif1b, Ttc38, Phyh, Mfsd6, LOC257642, Fam189b, Sned1, LOC100362849, Cd3d, Slc25a29, Cd7, RGD1563281, Grb7, Ccdc147, Stat2, LOC688228, Ppp3ca, LOC688241, Shisa4, Atp8b1, Krtap1-5, Cd3g, Txnl4b, Ranbp10, Dynlt3, Atp2b2, Trub1, Zhx3, Rexo4, Snapc2, LOC685031, Rad541, Vps37c, Hmga1, Olr1572, Furin, RT1-CE5, Rab15, Folr1, Fundc1, Pak3, Aqp2, Hapln2, Tmem37, Gjd3, Lamb1, Cox5a, Cfd, Jak3, Magt1, Sc4mol, Uros, Calca, Abca7, Pm20d2, Dpf2, Tmprss4, Znf467, Btbd9, Rasgrf1, RGD1310270, Adam15, Kif15, Plekho2, Atp2c2, Rps25, Lipa, Cpeb2, Znf575, LOC287010, Arhgap26, LOC311352, Znrf4, Msh5, Tmed7, Gpd1l, Ankrd37, Cox4i2, Msl3l2, Snrnp70, Rps6ka5, Tomm20, Rassf4, Hes1, Ctdsp1, Pomc, Inpp4b, Nfix, Ydjc, Cst3, Tesk2, Tcn2, RGD1309847, Artn, Wibg, Prpf4, LOC685179, Igsf3, Got1, Lcp2, Agrn, Cited2, Bat4, RGD1308059, Sft2d1, Ubac2, Scly, Wrb, Zfp161, Fads2, Fbxo8, RGD1563148, Vegfb, Lpl, Chaf1a, Srrd, Steap1, Fkbp11, Slc39a3, Zfp53, Bcan, Foxc2, Jph1, Ccl27, Pdia3, Gabrq, Surf6, Runx3, Abcb6, Cdk2, LOC687813, and Porcn.

In a preferred embodiment of the method for producing a composition for oral ingestion, the composition is a food for infants or a food for little children.

In a preferred embodiment of the method for producing a composition for oral ingestion, the food for infants or the food for little children is infant formula or follow-on formula.

MODES FOR CARRYING OUT THE INVENTION

The method of the present invention is a method for screening for a diet or a substance providing production of breast milk having an immunoregulatory action, which comprises identifying a diet or a substance that increases or decreases amount of mRNA present in milk of a mammal by using correlation of mRNA profile in the milk and a diet ingested by the mammal or a substance contained in the diet as an index.

In an embodiment of the aforementioned method, the immunoregulatory action is an immunostimulating action, and when either one or both of the following conditions are satisfied, it is determined that the diet or the substance provides production of breast milk having an immunostimulating action:

(A) the mRNA is a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet increases amount of the mRNA,

(B) the mRNA is a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet decreases amount of the mRNA.

In another embodiment of the aforementioned method, the immunoregulatory action is an immunosuppressive action, and when either one or both of the following conditions are satisfied, it is determined that the diet or the substance provides production of breast milk having an immunosuppressive action:

(a) the mRNA is a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet decreases amount of the mRNA,

(b) the mRNA is a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, and ingestion of the diet increases amount of the mRNA.

The present invention is based on a concept that an immunoregulatory action is expected to be obtained by oral administration of mRNA, because of the novel finding that mRNAs relating to immunoregulation are contained in milk, and the fact that breast milk promotes development of immunity in infants ingesting the breast milk (for example, Breastfeed Med., 2(4):195-204, 2007). And, on the basis of a prediction that mRNA profile in milk is affected by diet, it was thought to identify a diet or an active ingredient contained in it that could increase or decrease amount of mRNA present in milk.

The immunoregulatory action defined for the screening method, milk, dairy product, and so forth of the present invention includes, for example, both an action of enhancing immunopotentiating action, which functions for the purpose of “defense” against external attacks (immunostimulating action), and an immunosuppressive action suppressively functioning against overresponse by the immunity, i.e., allergic responses, autoimmune diseases, chronic inflammations etc., in which “hyperimmunoreaction” adversely affect living organisms.

The terms “immunostimulating action” and “immunosuppressive action” are used in a relative meaning. When an immunopotentiating action usually observed for breast milk of a certain mammal is enhanced after ingestion of the diet or the substance, the breast milk has an immunostimulating action, and when the immunopotentiating action is decreased after ingestion of the diet or the substance, the breast milk has an immunosuppressive action. When the immunopotentiating action observed after ingestion of the diet or the substance by a mammal is enhanced compared with that observed before the ingestion, the breast milk of the mammal has an immunostimulating action, and when the immunopotentiating action is decreased compared with that observed before the ingestion, the breast milk has an immunosuppressive action.

The correlation of mRNA profile in milk of a mammal and a diet ingested by the mammal or a substance contained in the diet can be investigated, for example, as follows.

Milk is collected from a mammal that ingested a diet, and mRNA profile in the milk is examined.

The mammal is not particularly limited, and examples include human, bovine, water buffalo, goat, ovine, swine, ape, dog, cat, rat, mouse, hamster, guinea pig, and so forth. The mammal is preferably human or bovine.

In the present invention, the mRNA profile consists of type and amount of mRNA. The mRNA may consist of one kind of mRNA, or arbitrary two or more kinds of mRNAs. Type of mRNA is not particularly limited, so long as those existing in milk and involved in immunoregulation are chosen. As described above, it is known that breast milk promotes development of immunity in infants who ingest it (for example, Breastfeed Med., 2(4):195-204, 2007). Therefore, it is suggested that mRNAs contained in milk include those involved in immune functions. Further, as shown in the examples, it has been demonstrated that mRNAs contained in milk include those of which amount is increased by administration of a Bifidobacterium having an immunoregulatory action, and those of which amount is decreased by the same. This fact means that mRNAs contained in milk include those functioning in an immunostimulating manner, and those functioning in an immunosuppressive manner.

Type of mRNA is not particularly limited, so long as those existing in milk and involved in immunoregulation are chosen. Specific examples include, for example, mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, and mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat.

Examples of mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat include those mentioned in Tables 1 to 14 mentioned later.

Examples of mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat include those mentioned in Tables 15 to 22 mentioned later.

In this specification, mRNA is specified by a name of corresponding gene, i.e., the gene used as a template for transcription of the mRNA.

A certain gene and a protein encoded by it may have different sequences depending on type of organism or due to individual difference. Genes showing high sequence homology and coding for proteins having the same functions or analogous functions are often regarded as the same genes or analogous genes as homologues or orthologues. In the present invention, mRNAs derived from the same or analogous genes are regarded as the same mRNAs. Therefore, even when “mRNA” in the expression “mRNA profile in milk of a mammal” and corresponding “mRNA” of which amount in milk of a rat increases or decreases when a Bifidobacterium is orally administered to the rat have different sequences, they may be regarded as the same mRNA, so long as they show high sequence homology and code for proteins having the same or analogous functions.

mRNA is not limited to mRNA having a known sequence, but mRNA may contain substitution, deletion, insertion, addition, or inversion of one or several nucleotides, so long as the encoded protein has function the same as or analogous to that of a protein encoded by a known sequence. Specific examples of such mRNA include, for example, mRNA having a nucleotide sequence showing a homology of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, to a known mRNA.

The amount of mRNA may be an absolute amount or a relative amount. The relative amount may be a relative amount based on an average amount in a certain mammal, or may be a relative amount observed after ingestion of a diet based on the amount observed before the ingestion. For the measurement of the amount of mRNA, methods usually used for measurement of mRNA amount such as quantitative reverse transcription PCR (qRT-PCR) can be employed. The amount of mRNA can also be measured by the microarray method. As for extraction of mRNA from milk, methods usually used for extraction of mRNA can be employed, and a commercially available mRNA isolation kit can also be used.

Further, amount of mRNA present in milk can also be indirectly measured by measuring expression amount of the mRNA in mammary glandular cells.

Correlation of mRNA profile in milk of a mammal and a diet ingested by the mammal or a substance contained in the diet is examined. The correlation of the mRNA profile in milk of a mammal and a diet ingested by the mammal or a substance contained in the diet refers to correlation of the mRNA profile and presence or absence of the diet or the substance or amount of the diet or the substance. For example, if amounts of one or more kinds of mRNAs in milk of a mammal which has ingested a certain diet or substance are higher or lower than those observed in the mammal which has not ingested the diet or the substance, the diet or the substance and the mRNA profile have positive or negative correlation, respectively. Further, also when amounts of one or more kinds of mRNAs in milk of a mammal which has ingested a certain diet or substance are higher than those observed in the mammal which has not ingested the diet or the substance, and at the same time, amounts of other one or more kinds of mRNAs in milk of a mammal which has ingested a certain diet or substance are lower than those observed in the mammal which has not ingested the diet or the substance, the diet or the substance and the mRNA profile have correlation. Further, if ingestion of a certain diet or substance does not affect mRNA profile, the substance and the mRNA profile do not correlate with each other.

Specifically, for example, when mRNA profiles in the milk observed before and after ingestion of the diet are compared, and amount of a certain kind of mRNA observed after the ingestion is higher than that observed before the ingestion, it is determined that the diet increases the amount of the mRNA in the milk. The degree of increase in amount of mRNA in the milk observed after the ingestion of the diet is, for example, 1.5 times or more, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more, of that observed before the ingestion.

Further, when mRNA profiles in the milk observed before and after ingestion of the diet are compared, and amount of a certain kind of mRNA observed after the ingestion is lower than that observed before the ingestion, it is determined that the diet decreases the amount of the mRNA in the milk. The degree of decrease in amount of mRNA in the milk observed after the ingestion of the diet is, for example, 0.67 times or less, preferably 0.5 times or less, more preferably 0.2 times or less, still more preferably 0.1 times or less, of that observed before the ingestion.

Furthermore, measurement of mRNA profile before ingestion of a diet is not indispensable, and correlation of a diet and amount of mRNA can also be examined by comparing mRNA profile measured after ingestion of a diet with ordinary mRNA profile of an objective mammal measured beforehand.

mRNA of which correlation is investigated may consist of a single kind of mRNA, or arbitrary two or more kinds of mRNAs.

In another embodiment, mRNA profile in milk and mRNA profile in serum or plasma are compared, and if amount of mRNA contained in both of milk and serum or plasma is increased by ingestion of the diet at a higher degree in milk compared with that observed in serum or plasma, it is determined that the diet increases amount of the mRNA present in milk. The degree of increase in amount of mRNA in milk is, for example, 1.5 times or more, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more, of that observed in serum or plasma.

Further, when mRNA profile in milk and mRNA profile in serum or plasma are compared, if amount of a mRNA contained in both of milk and serum or plasma is decreased by ingestion of the diet at a larger degree in milk compared with that observed in serum or plasma, it is determined that the diet decreases amount of the mRNA present in milk. The degree of decrease in amount of mRNA in milk compared with that in serum or plasma is, for example, 1.5 times or more, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more. For example, when amount of a certain mRNA decreases to 0.5 times in serum or plasma and decreases to 0.3 times in milk, degree of the decrease in milk is 1.67 (0.5/0.3) times higher than that in serum or plasma.

In addition, when there is a factor that increases or decreases amount of mRNA in milk, and increase or decrease of the amount of the mRNA is suppressed by ingestion of a certain diet, the diet has an action of decreasing or increasing the amount of the mRNA.

The diet may consist of a single substance or may be a composition, so long as it can be orally ingested. Further, “before ingestion” and “after ingestion” may mean “before and after one time of ingestion of diet”, or “before and after two or more times of ingestion of diet”. Further, two or more times of ingestion of diet may be two or more times of ingestion of the same diet, or ingestion of two or more kinds of diets.

The diet may be ingested according to a planned scheme or freely ingested. In the latter case, correlation of the diet and mRNA profile in milk can be examined by hearing content of ingested diet in the case of human. When the diet is ingested or administered according to a planned scheme, the diet can be considered as a “test sample”. The diet may be a usual diet or a usual diet containing a test substance. Amount of diet to be ingested, time of ingestion, number of times of ingestion and so forth are not particularly limited.

If a diet that increases amount of mRNA in milk is chosen, a substance that is contained in the diet and increases amount of the mRNA in milk can be identified in the same manner as that mentioned above. Further, if a diet that decreases amount of mRNA in milk is chosen, a substance that is contained in the diet and decreases amount of the mRNA in milk can be identified in the same manner as that mentioned above.

If a diet or a substance that increases or decreases amount of mRNA in milk is identified, a diet that increases or decreases amount of the mRNA in milk can be designed.

That is, a diet or a substance contained in the diet of which ingestion increases amount of mRNA of the same kind as that of mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, among mRNAs in milk, is preferred for production of milk having an immunostimulating action. Further, a diet or a substance contained in the diet of which ingestion decreases amount of mRNA of the same kind as that of mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, among mRNAs in milk, is also preferred for production of milk having an immunostimulating action. Further, it is considered that these diets or substances are unsuitable for production of milk having an immunosuppressive action.

On the other hand, a diet or a substance contained in the diet of which ingestion decreases amount of mRNA of the same kind as that of mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, among mRNAs in milk, is preferred for production of milk having an immunosuppressive action. Further, a diet or a substance contained in the diet of which ingestion increases amount of mRNA of the same kind as that of mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, among mRNAs in milk, is also preferred for production of milk having an immunosuppressive action. Further, it is considered that these diets or substances are unsuitable for production of milk having an immunostimulating action.

Screening for a diet or a substance providing production of breast milk having an immunoregulatory action, or a diet or a substance unsuitable for production of breast milk having an immunoregulatory action can be performed as described above. As shown in the examples described later, when a Bifidobacterium (Bifidobacterium longum) was orally administered to rats, amounts of 562 kinds of mRNAs increased, and amounts of 325 kinds of mRNAs decreased.

It is known that Bifidobacteria function as probiotics, and have, in particular, an immunoregulatory action. Therefore, the fact that the administration of the Bifidobacterium increased amounts of mRNAs in milk also supports the involvement of mRNAs in milk in immunoregulation. Demonstration of change in amounts of mRNAs in milk induced by administration of the Bifidobacterium, i.e., correlation of the Bifidobacterium and mRNA profile, shows that the screening method of the present invention is feasible. Further, although there were also mRNAs of which amounts in milk were not changed by administration of the Bifidobacterium, a possibility that amounts of those mRNAs may be increased or decreased by another kind of diet or a substance contained therein is not denied.

As probiotic functions of Bifidobacteria, there are known prophylaxis or amelioration of respiratory tract infection, acute infectious diarrhea, antibiotic-associated diarrhea, Clostridium dificile-associated diarrhea, necrotizing enterocolitis, traveler's diarrhea, Helicobacter pylori infection, and so forth (The Journal of Nutrition, 2010 March; 140(3):698S-712S. Epub 2010 Jan. 27). It is suggested that mRNA of which amount in milk is increased by administration of Bifidobacteria not only regulates immunity, but also exhibits functions similar to the aforementioned probiotic functions in mammals that ingested them.

By giving a diet or a substance that increases amount of mRNA in milk functioning in an immunostimulating manner chosen as described above to a mammal, and collecting milk from the mammal, milk having an immunostimulating action or milk of which immunostimulating action is enhanced can be obtained. Further, by reducing or avoiding ingestion by a mammal of a diet or a substance that decreases amount of mRNA in milk functioning in an immunostimulating manner chosen as described above, an immunostimulating action of milk can be enhanced, or decrease of an immunostimulating action can be prevented. Ingestion of a diet or a substance that increases or decreases amount of mRNA in milk, and reduction or avoidance of ingestion of a diet or a substance that decreases or increases amount of mRNA in milk may be used in combination.

Further, by giving a diet or a substance that increases amount of mRNA in milk functioning in an immunosuppressive manner chosen as described above to a mammal, and collecting milk from the mammal, milk having an immunosuppressive action or milk of which immunostimulating action is decreased can be obtained. Further, by reducing or avoiding ingestion by a mammal of a diet or a substance that decreases amount of mRNA in milk functioning in an immunosuppressive manner chosen as described above, an immunosuppressive action of milk can be enhanced, or decrease of an immunosuppressive action can be prevented. Ingestion of a diet or a substance that decreases or increases amount of mRNA in milk, and reduction or avoidance of ingestion of a diet or a substance that increases or decreases amount of mRNA in milk may be used in combination.

By processing milk having an immunoregulatory action obtained as described above, a dairy product having an immunoregulatory action can be produced.

Type of the dairy product is not particularly limited, so long as mRNA can exist in it with maintaining the functions thereof, and examples include processed milk, infant formula, milk beverages, powdered infant formula, fermented milk, cream, butter, cheese, ice cream, and so forth. As the dairy product, a dairy product for infants or little children is preferred.

According to the present invention, there was demonstrated presence in milk of mRNAs, especially mRNAs which have been known to participate in enhancement of immunity, such as development of immunity, antiallergy, anti-inflammation, and defense against infection. In addition, it is well known that breast milk gives an immunostimulating action to an infant who ingested it. Therefore, it is rationally predicted that mRNA participating in immunoregulation can regulate immunity of organism such as human who ingested it. Therefore, it is considered that transfer of various mRNAs from a mother to an infant is extremely significant for, in particular, infants having an underdeveloped immune system.

Another aspect of the present invention is a method for producing a composition for oral ingestion having an immunostimulating action, which comprises adding a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat to a base for a composition for oral ingestion. Examples of the mRNA include those mentioned in Tables 1 to 14 mentioned later.

Still another aspect of the present invention is a method for producing a composition for oral ingestion having an immunosuppressive action, which comprises adding a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat to a base for a composition for oral ingestion. Examples of the mRNA include those mentioned in Tables 15 to 22 mentioned later.

The mRNA may consist of a single kind of mRNA or arbitrary two or more kinds of mRNAs.

The base for composition for oral ingestion is not particularly limited so long as an orally ingestible or administrable base in which mRNA can exist with maintaining functions thereof is chosen, and examples include foods, drinks, drug bases, feeds, and so forth.

Foods may be in any form, and include drinks. Foods include foods for adults, foods for infants, foods for little children, and so forth.

Examples of the foods for adults include enteral nutrients, fluid diets such as concentrated fluid diets, nutritional supplementary foods, and so forth.

Examples of the foods for infants or the foods for little children include, for example, modified milks (for example, infant formula, infant formula for low birth weight infants, follow-on formula, etc. as well as infant formula for allergic infants, non-lactose milk, special milk for inborn errors of metabolism infants, etc., and dried milk prepared from these), powders for supplement of breast milk or powdered infant formula, baby food, and so forth.

The infant formula referred to here are foods produced by using milk or dairy products as main raw materials, and adding nutrients required for infants, and are mainly used as alternative food for breast milk in infancy, and as alternative food for breast milk or nutritional complementary food in childhood. Other examples thereof include foods produced for the purpose of contributing to nutritional ingestion suitable for infants with a specific inherent or acquired disease.

mRNA is relatively resistant to freeze-thaw, low pH such as acidic condition of pH 2, and RNases (Hata, T. et al., Biochem. Biophys. Res. Commun., 396:528-533, 2010), and thus is suitable as an active ingredient to be added to foods. The stability at a low pH suggests that mRNA molecules are resistant to the infant's intragastric environment, and can be absorbed by the intestinal tract, which is one of the main immune organs of infants, and thus they can affect the immune system of infants. Further, storage and freeze-thaw of breast milk do not denature mRNA, and this is nutritionally important for low birth weight infants and hospitalized infants, who are usually given cryopreserved breast milk. Furthermore, the resistance of mRNA to RNases suggests that mRNA may exist in a complex such as exosome and microvesicle in breast milk. Similarly, when mRNA is added to a composition for oral ingestion, mRNA may be micellized by using, for example, lipid bilayer or the like.

Although content of mRNA in the composition for oral ingestion is not particularly limited, and may be appropriately chosen, it is, for example, 10 to 10,000 ng/ml, preferably 20 to 10,000 ng/ml, more preferably 50 to 10,000 ng/ml, in total. Further, amount of mRNA to be ingested is, for example, 5 μg to 120 mg/day, preferably 10 μg to 120 mg/day, more preferably 25 μg to 120 mg/day, in total.

mRNA can be prepared by, for example, gene engineering using a cell introduced with an expression vector that expresses a corresponding gene.

Further, mRNA can also be prepared by chemical synthesis. That is, mRNA can be obtained by synthesizing a sense strand and an antisense strand and annealing them.

Further, mRNA can also be prepared from breast milk or a fractionation product thereof containing mRNA. For example, mRNA that functions in an immunostimulating manner, for example, a mRNA of which amount in milk of a rat increases when a Bifidobacterium is orally administered to the rat, can be collected from breast milk of a mammal given a diet or a substance that increases amount of the mRNA in milk. Further, mRNA that functions in an immunosuppressive manner, for example, a mRNA of which amount in milk of a rat decreases when a Bifidobacterium is orally administered to the rat, can be collected from breast milk of a mammal given a diet or a substance that increases amount of the mRNA in milk. mRNA may be a purified product or a crude product. mRNA that functions in an immunostimulating manner may contain a small amount of mRNA that functions in an immunosuppressive manner, so long as the immunostimulating action of the mRNA is not degraded. Further, mRNA that functions in an immunosuppressive manner may contain a small amount of mRNA that functions in an immunostimulating manner, so long as the immunosuppressive action of the mRNA is not degraded.

When the composition for oral ingestion of the present invention is a pharmaceutical agent, the composition can be prepared by combining mRNA with pharmaceutically acceptable carriers for oral administration. The form of the pharmaceutical preparation is not particularly limited, and examples include tablet, pill, powder, solution, suspension, emulsion, granule, capsule, syrup, and so forth. For the formulation, additives widely used for usual pharmaceutical agents as pharmaceutical carriers for oral administration such as excipients, binders, disintegrating agents, lubricants, stabilizers, corrigents, diluents, and surfactants can be used. Further, unless the effect of the present invention is degraded, mRNA may be used together with another drug having an immunoregulatory action.

Although amount of mRNA contained in the pharmaceutical agent is not particularly limited, and can be appropriately chosen, it is, for example, 2 μg/kg to 2 mg/kg, preferably 4 μg/kg to 2 mg/kg, more preferably 10 μg/kg to 2 mg/kg, in total.

When the composition for oral ingestion is a food, it may be for any of various uses utilizing an immunostimulating action. Examples of the use include, for example, uses as foods suitable for persons showing reduced resistance, uses as foods or drinks useful for reduction and elimination of risk factors of various diseases caused by immune depression, and so forth.

The foods or drinks of the present invention can be marketed as foods attached with an indication describing that the foods or drinks are used for immunoregulation.

The aforementioned term “indication” includes all actions for informing consumers the aforementioned use, and any indications reminding or analogizing the aforementioned use fall within the scope of the “indication” of the present invention regardless of purpose, content, objective article, medium etc. of the indication. However, the indication is preferably made with an expression that allows consumers to directly recognize the aforementioned use. Specific examples include actions of indicating the aforementioned use on goods or packages of goods relating to the food of the present invention, actions of assigning, delivering, displaying for the purpose of assigning or delivering or importing such goods or packages of goods on which the aforementioned use is indicated, displaying or distributing advertisements, price lists or business papers relating the goods on which the aforementioned use is indicated, or providing information including those as contents with indicating the aforementioned use by an electromagnetic method (Internet etc.) and so forth.

The indication is preferably an indication approved by the administration etc. (for example, an indication in a form based on an approval, which is qualified on the basis of any of various legal systems provided by the administration), and it is particularly preferably an indication on advertisement materials at the sales spots such as packages, containers, catalogs, pamphlets and POPs (point of purchase advertising), other documents and so forth.

Examples of the indication further include, for example, indications as health food, functional food, enteric nutritive food, food for special dietary uses, food with nutrient function claims, quasi-drug and so forth as well as indications approved by the Ministry of Health, Labor and Welfare, for example, indications approved on the basis of the system of food for specified health uses and similar systems. Examples of the latter include indications as food for specified health uses, indications as food for specified health uses with qualified health claims, indications of influence on body structures and functions, indications of reduction of disease risk claims and so forth, and more precisely, typical examples include indications as food for specified health uses (especially indications of use for health) provided in the enforcement regulations of Health Promotion Law (Japanese Ministry of Health, Labor and Welfare, Ministerial ordinance No. 86, Apr. 30, 2003) and similar indications.

As shown in the examples, rat milk contained many kinds of mRNAs derived from genes coding for hormones such as growth hormone, cholecystokinin, and gastrin, growth factors such as epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, and vascular endothelial growth factor, enzymes such as fatty acid synthase, lipoprotein lipase, and acyl-CoA synthase, and so forth. It is considered that these proteins have an action of promoting growth of children (baby mammals). Therefore, the present invention can be similarly applied to screening for a diet or a substance that provides production of breast milk having an enhanced action of promoting growth of children (baby mammals), a method for producing milk or a dairy product having such an action, and a method for producing a composition for oral ingestion having such an action.

EXAMPLES

Hereafter, the present invention will be further specifically explained with reference to examples. However, the present invention is not limited to the following examples.

Example 1

Analysis of mRNA in Rat Milk

Three F344 rats on day 14 of gestation were purchased. All the purchased rats gave birth on day 21 of gestation, and they were milked 2, 9 and 16 days after the delivery under ether anesthesia to collect milk. Further, they were dissected 21 days after the delivery, which corresponded to the weaning day, and blood was collected from the inferior vena cava.

Each milk sample was centrifuged at 1,200×g and 4° C. for 10 minutes, and the lipid layer and cell debris were removed.

Then, each sample was centrifuged twice at 21,500×g and 4° C. for 30 minutes, further centrifuged once under the same conditions for 1 hour to remove the casein fraction, and passed through membranes of 0.65 μm, 0.45 μm and 0.22 μm to remove cell debris and insoluble matter and thereby obtain a milk serum sample.

The blood obtained by the blood collection was centrifuged at 1,200×g and 4° C. for 10 minutes, and passed through membranes of 0.65 μm, 0.45 μm and 0.22 μm to remove cell debris and insoluble matter and thereby obtain a blood serum sample.

Total RNAs were obtained from the obtained milk serum sample and the blood serum sample by using miRNeasy Mini Kit (Qiagen). Since the blood serum sample contained only a small amount of RNA, the blood sera of three rats were combined, and the total RNA was obtained from the combined blood serum.

After quality of each obtained RNA solution was confirmed by using BioAnalyzer, 100 ng of RNA was used to synthesize cRNA by using Low RNA Input Liner Amplification Kit (Agilent Technologies). At the time of this synthesis, CPT labeled with a cyanine dye (Cyanine 3-CPT) was incorporated into cRNA to attain fluorescence labeling of cRNA. The labeled cRNA was added to a hybridization buffer, allowed to hybridize on Whole Rat Genome Oligo Microarray (4×44 K, Agilent Technologies) for 17 hours, washed, and then imaged with Agilent Microarray Scanner (Agilent Technologies), and the fluorescent signals of the spots were numerically represented by using Feature Extraction Software. The results were analyzed by using GeneSpring GX11.0 (Agilent Technologies).

As a result, in the milk serum sample, mRNAs corresponding to 10,948 kinds of probes were detected, and in the blood serum sample, mRNAs corresponding to 2,361 kinds of probes were detected. The mRNAs detected in the milk serum sample included many mRNAs derived from genes coding for proteins relating to the immune system, such as cytokines, chemokines, and antimicrobial peptides. In fact, when biological interpretation was performed by using Ingenuity Pathway Analysis (Ingenuity Systems), it was demonstrated that many mRNAs relating to pathways of “infection mechanism” or “infectious disease” were included. An action of defending children (baby mammals) from infection is known for milk, and interestingly, the defense from infection has also been suggested on the mRNA level. In addition, many mRNAs derived from genes coding for proteins that promote growth of children (baby mammals) such as hormones, growth factors, and enzymes were also contained.

Example 2

Identification of Diet or Substance Providing Production of Milk Having Immunoregulatory Action

SD rats on day 9 to 10 of gestation were purchased, and during the period from day 13 to day 20 of gestation, a suspension in which cells of a Bifidobacterium, Bifidobacterium longum BB536 (ATCC BAA-999), were suspended in PBS (phosphate buffered saline) (1×10⁹ cfu/ml) was orally administered everyday to the rats of test group (n=3) in a volume of 1 ml/day per rat.

Further, 1 ml/day per rat of PBS was administered everyday to the rats of control group (n=3). The B. longum ATCC BAA-999 strain can be purchased from American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Md. 20852, United States of America).

All the rats gave birth on day 21 of gestation, and they were milked under ether anesthesia one day after the birth. Each obtained milk sample was centrifuged at 1,200×g and 4° C. for 10 minutes, and the lipid layer and cell debris were removed.

Then, each sample was centrifuged twice at 21,500×g and 4° C. for 30 minutes, further centrifuged once under the same conditions for 1 hour to remove the casein fraction, and passed through membranes of 0.65 μm, 0.45 μm and 0.22 μm to remove cell debris and insoluble matter and thereby obtain a milk serum sample.

Total RNA was obtained from the obtained milk serum sample by using miRNeasy Mini Kit (Qiagen). By using 100 ng of the obtained RNA sample, detection of mRNAs and analysis of expression amounts thereof were conducted in the same manner as that of Example 1.

As a result, number of probes corresponding to mRNAs confirmed in the test group and the control group was 10,239.

Further, the expression amounts in the Bifidobacterium longum BB536 strain-administered group and the control group were compared, and evaluated by the Mann-Whitney U-test. As a result, at a probability level of P<0.05, number of mRNAs that increased 1.5 times or more was 562, and number of mRNAs that decreased to 0.67 times or less was 325. These mRNAs and rate of change (RC) in expression amounts thereof are shown in Tables 1 to 22. The mRNAs of which expression amounts increased are mentioned in Tables 1 to 14, and the mRNAs of which expression amounts decreased are mentioned in Tables 15 to 22.

TABLE 1

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P235892

7.30

Cyp24a1

25279

cytochrome P450, family 24, subfamily a, polypeptide 1

A_44_P468182

6.80

Dnm1

140694

dynamin 1

A_44_P311647

6.64

Dpysl2

25416

dihydropyrimidinase-like 2

A_42_P619403

6.47

Fam89a

361441

family with sequence similarity 89, member A

A_44_P1041539

6.05

Arhgap22

306279

Rho GTPase activating protein 22

A_43_P17699

5.86

Fbxl5

305424

F-box and leucine-rich repeat protein 5

A_44_P220008

5.85

LOC304396

304396

similar to hypothetical protein DKFZp434K1815

A_44_P398142

5.40

Btg2

29619

BTG family, member 2

A_44_P253782

5.29

Csnk1g1

64086

casein kinase 1, gamma 1

A_44_P607813

5.19

Mll1

315606

myeloid/lymphoid or mixed-lineage leukemia 1

A_44_P496025

4.78

Eml4

313861

echinoderm microtubule associated protein like 4

A_44_P494147

4.76

Nelf

117536

nasal embryonic LHRH factor

A_43_P12247

4.74

Aldh1l1

64392

aldehyde dehydrogenase 1 family, member L1

A_44_P392004

4.68

Boc

360715

biregional cell adhesion molecule-related/down-regulated by

oncogenes (Cdon) binding protein

A_44_P347420

4.67

Usp43_predicted

100147704

ubiquitin specific protease 43

A_44_P442802

4.60

Rassf5

54355

Ras association (RalGDS/AF-6) domain family member 5

A_44_P1038694

4.59

Man2a1

25478

mannosidase, alpha, class 2A, member 1

A_42_P559737

4.42

Srgn

56782

serglycin

A_43_P10254

4.41

Gbf1

309451

golgi-specific brefeldin A resistant guanine nucleotide

exchange factor 1

A_44_P869362

4.38

RGD1562551

311760

similar to C20orf82

A_43_P18573

4.37

Lrba

361975

LPS-responsive vesicle trafficking, beach and anchor

containing

A_44_P520159

4.34

P2ry2

29597

purinergic receptor P2Y, G-protein coupled, 2

A_42_P623909

4.21

Bcl2l1

24888

Bcl2-like 1

A_44_P251673

4.18

Rbm14

170900

RNA binding motif protein 14

A_44_P242339

4.13

RGD1307365

300880

similar to KIAA1009 protein

A_43_P21138

4.11

Tecpr1

304285

tectonin beta-propeller repeat containing 1

A_43_P10399

4.11

Kctd9

364410

potassium channel tetramerisation domain containing 9

A_44_P545478

4.10

Pkig

266709

protein kinase inhibitor, gamma

A_43_P19970

4.09

RGD1308139

360563

similar to RIKEN cDNA 1200014J11

A_43_P22368

4.05

Klhl35

308850

kelch-like 35 (Drosophila)

A_44_P176342

4.03

Gab1

361388

GRB2-associated binding protein 1

A_44_P253196

4.02

Arid5b

309728

AT rich interactive domain 5B (Mrf1 like)

A_44_P871726

4.01

Zfp157

360775

zinc finger protein 157

A_43_P10581

4.00

Mafk

246760

v-maf musculoaponeurotic fibrosarcoma oncogene homolog K

(avian)

A_42_P700288

3.93

Trim47

690374

tripartite motif-containing 47

A_44_P471166

3.89

Ddx21

317399

DEAD (Asp-Glu-Ala-Asp) box polypeptide 21

A_44_P471018

3.86

Tiparp

310467

TCDD-inducible poly(ADP-ribose) polymerase

A_44_P1042876

3.86

Hspa1b

294254

heat shock 70 kD protein 1B (mapped)

A_44_P977318

3.80

Tcfcp2l1

304741

transcription factor CP2-like 1

A_43_P15281

3.78

Nr3c2

25672

nuclear receptor subfamily 3, group C, member 2

A_43_P12908

3.76

Klf4

114505

Kruppel-like factor 4 (gut)

TABLE 2

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P547957

3.76

Dhx8

287727

DEAH (Asp-Glu-Ala-His) box polypeptide 8

A_44_P140092

3.75

Myh14

308572

myosin, heavy chain 14

A_44_P317388

3.73

Mcam

78967

melanoma cell adhesion molecule

A_44_P729514

3.71

Dusp3

498003

dual specificity phosphatase 3

A_44_P674018

3.71

Acer2

313339

alkaline ceramidase 2

A_44_P668591

3.70

L3mbtl3

309550

l(3)mbt-like 3 (Drosophila)

A_44_P468489

3.68

Fcho2

309129

FCH domain only 2

A_42_P521707

3.66

RT1-N3

24750

RT1 class Ib, locus N3

A_44_P879104

3.63

Ash1l

310638

ash1 (absent, small, or homeotic)-like (Drosophila)

A_44_P294467

3.59

Paxip1

311944

PAX interacting (with transcription-activation domain) protein 1

A_44_P170778

3.57

Irgq

292708

immunity-related GTPase family, Q

A_44_P199211

3.55

Plcb1

24654

phospholipase C, beta 1 (phosphoinositide-specific)

A_42_P702550

3.54

Sts

24800

steroid sulfatase

A_44_P166068

3.54

Spetex-2D

364260

Spetex-2D protein

A_44_P389462

3.54

Zmym3

317260

zinc finger, MYM-type 3

A_43_P11614

3.49

Anxa1

25380

annexin A1

A_44_P384677

3.45

Rtn4ip1

309912

reticulon 4 interacting protein 1

A_44_P254366

3.43

Gngt1

680149

guanine nucleotide binding protein (G protein), gamma

transducing activity polypeptide 1

A_42_P615851

3.43

Srcap

361652

Snf2-related CREBBP activator protein

A_44_P213208

3.41

Otud4

307774

OTU domain containing 4

A_43_P17271

3.38

RGD1307569

360695

similar to Protein C21orf63 homolog precursor

A_43_P21650

3.36

Adpgk

315722

ADP-dependent glucokinase

A_44_P135224

3.36

Plk3

58936

polo-like kinase 3 (Drosophila)

A_44_P1057137

3.34

Phf1

294287

PHD finger protein 1

A_43_P12878

3.33

St14

114093

suppression of tumorigenicity 14 (colon carcinoma)

A_44_P1044517

3.32

N4bp1

291921

Nedd4 binding protein 1

A_43_P17298

3.30

Naaa

497009

N-acylethanolamine acid amidase

A_43_P13203

3.29

Expi

171059

extracellular proteinase inhibitor

A_44_P392234

3.28

Fgf7

29348

fibroblast growth factor 7

A_44_P651749

3.26

RGD1562492

499151

similar to Orphan sodium- and chloride-dependent

neurotransmitter transporter NTT5 (Solute carrier family 6

member 16)

A_43_P21455

3.26

Lama2

309368

laminin, alpha 2

A_44_P342075

3.25

Ahnak

191572

AHNAK nucleoprotein

A_44_P414757

3.25

Sec3l1

305287

SEC3-like 1 (S. cerevisiae)

A_44_P635769

3.25

Mks1

287612

Meckel syndrome, type 1

A_43_P16750

3.24

Prodh

680409

proline dehydrogenase

A_42_P831092

3.23

Baz1b

368002

bromodomain adjacent to zinc finger domain, 1B

A_42_P658005

3.21

R3hdm1

304763

R3H domain containing 1

A_44_P869639

3.20

Setd5

297514

SET domain containing 5

A_44_P1050325

3.20

Rhebl1

359959

Ras homolog enriched in brain like 1

A_44_P175461

3.19

Kdm5b

304809

lysine (K)-specific demethylase 5B

A_43_P18798

3.19

Fbxw8

304522

F-box and WD repeat domain containing 8

A_42_P625263

3.19

Epb4.1l4a

307514

erythrocyte protein band 4.1-like 4a

A_44_P690201

3.15

Lpin2

316737

lipin 2

A_44_P515197

3.14

Cxcl2

114105

chemokine (C-X-C motif) ligand 2

TABLE 3

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P522388

3.14

Nr2c2

50659

nuclear receptor subfamily 2, group C, member 2

A_44_P408050

3.11

Ckap4

362859

cytoskeleton-associated protein 4

A_44_P284753

3.10

Ankrd1

27064

ankyrin repeat domain 1 (cardiac muscle)

A_44_P172604

3.09

Ugcg

83626

UDP-glucose ceramide glucosyltransferase

A_42_P735417

3.08

Fcgr2b

289211

Fc fragment of IgG, low affinity IIb, receptor (CD32)

A_44_P668137

3.07

Sf3a1

305479

splicing factor 3a, subunit 1

A_44_P503570

3.07

Parg

83507

poly (ADP-ribose) glycohydrolase

A_44_P905278

3.06

Heatr6

497972

HEAT repeat containing 6

A_42_P671285

3.06

Ctdspl

301056

CTD (carboxy-terminal domain, RNA polymerase II,

polypeptide A) small phosphatase-like

A_44_P276782

3.04

Tp53bp1

296099

tumor protein p53 binding protein 1

A_42_P772136

3.02

Fam60a

686611

family with sequence similarity 60, member A

A_43_P12150

3.02

Pik3r2

29741

phosphoinositide-3-kinase, regulatory subunit 2 (beta)

A_44_P157078

3.00

RGD1305089

299050

similar to 1110008L16Rik protein

A_44_P930917

3.00

Arhgef5

140898

Rho guanine nucleotide exchange factor (GEF) 5

A_44_P281155

2.98

Nek7

360850

NIMA (never in mitosis gene a)-related kinase 7

A_44_P926632

2.95

Rp2

367714

retinitis pigmentosa 2 homolog (human)

A_44_P477135

2.94

Vhl

24874

von Hippel-Lindau tumor suppressor

A_43_P17943

2.94

Usp31

308959

ubiquitin specific peptidase 31

A_44_P1011373

2.93

Camsap1

296580

calmodulin regulated spectrin-associated protein 1

A_44_P914747

2.93

RGD1308759

290668

similar to KIAA0892 protein

A_44_P854689

2.90

Ldlrap1

500564

low density lipoprotein receptor adaptor protein 1

A_44_P437641

2.89

RGD1359108

313155

similar to RIKEN cDNA 3110043O21

A_44_P681569

2.88

Sp1

24790

Sp1 transcription factor

A_44_P623197

2.88

Mast4

310040

microtubule associated serine/threonine kinase family member 4

A_44_P184726

2.88

Rela

309165

v-rel reticuloendotheliosis viral oncogene homolog A (avian)

A_44_P791631

2.88

Nhlrc3

310416

NHL repeat containing 3

A_43_P22909

2.87

Intu

361938

inturned planar cell polarity effector homolog (Drosophila)

A_44_P138800

2.86

Gcap14

306306

granule cell antiserum positive 14

A_44_P185015

2.84

Spetex-2B

497084

Spetex-2B protein

A_44_P959960

2.84

Cdc42se2

691031

CDC42 small effector 2

A_44_P485388

2.83

Chd7

312974

chromodomain helicase DNA binding protein 7

A_44_P306964

2.83

Rbms1

362138

RNA binding motif, single stranded interacting protein 1

A_44_P624151

2.82

Invs

313228

inversin

A_44_P321488

2.82

LOC100294508

100294508

dyslexia susceptibility 2-like

A_44_P989302

2.81

Kif2a

84391

kinesin family member 2A

A_44_P401515

2.81

Exoc6

50556

exocyst complex component 6

A_43_P18884

2.81

Zbtb1

314246

zinc finger and BTB domain containing 1

A_44_P414460

2.80

Rnd3

295588

Rho family GTPase 3

A_44_P122401

2.79

Rb1cc1

312927

RB1-inducible coiled-coil 1

A_44_P381917

2.78

Sphk1

170897

sphingosine kinase 1

TABLE 4

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P402641

2.78

Ugt1a2

396527

UDP glucuronosyltransferase 1 family, polypeptide A2

A_44_P401131

2.76

Noxo1

302976

NADPH oxidase organizer 1

A_44_P173657

2.76

Mllt6

303504

myeloid/lymphoid or mixed-lineage leukemia (trithorax

homolog, Drosophila); translocated to, 6

A_44_P102001

2.76

Thumpd2

313851

THUMP domain containing 2

A_44_P194172

2.74

Ptpn4

246116

protein tyrosine phosphatase, non-receptor type 4

A_44_P1060513

2.74

Thex1

361159

three prime histone mRNA exoribonuclease 1

A_44_P874704

2.74

S100a8

116547

S100 calcium binding protein A8

A_44_P363229

2.71

Grk5

59075

G protein-coupled receptor kinase 5

A_44_P825195

2.71

Trove2

304833

TROVE domain family, member 2

A_44_P684740

2.70

Pcdh17

306055

protocadherin 17

A_44_P445972

2.70

Med13

303403

mediator complex subunit 13

A_44_P522047

2.70

LOC684871

684871

similar to Protein C8orf4 (Thyroid cancer protein 1) (TC-1)

A_44_P295467

2.69

RGD1305350

313699

similar to RIKEN cDNA 2510039O18

A_44_P365882

2.69

Fam82a2

311328

family with sequence similarity 82, member A2

A_44_P161602

2.68

Rab43

500249

RAB43, member RAS oncogene family

A_44_P1031034

2.67

Clip1

65201

CAP-GLY domain containing linker protein 1

A_44_P976816

2.66

Ankrd57

503306

ankyrin repeat domain 57

A_44_P344928

2.65

RGD1563375

366392

similar to small nuclear ribonucleoparticle-associated protein

A_42_P538084

2.64

Crim1

298744

cysteine rich transmembrane BMP regulator 1 (chordin like)

A_43_P15220

2.64

Celsr2

83465

cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo

homolog, Drosophila)

A_43_P12569

2.63

Idh1

24479

isocitrate dehydrogenase 1 (NADP+), soluble

A_44_P372148

2.63

Lrrc61

500111

leucine rich repeat containing 61

A_44_P1034030

2.63

Aff4

303132

AF4/FMR2 family, member 4

A_44_P520557

2.63

Arhgap1

311193

Rho GTPase activating protein 1

A_44_P288187

2.63

Itpr3

25679

inositol 1,4,5-triphosphate receptor, type 3

A_44_P440178

2.62

Lhfp

499615

lipoma HMGIC fusion partner

A_44_P1005649

2.62

Dnajc12

619393

DnaJ (Hsp40) homolog, subfamily C, member 12

A_44_P539680

2.62

Garnl1

56785

GTPase activating Rap/RanGAP domain-like 1

A_42_P521902

2.61

Gtf3c5

362095

general transcription factor IIIC, polypeptide 5

A_44_P134143

2.60

Hspb8

113906

heat shock protein B8

A_43_P17471

2.59

Chac1

362196

ChaC, cation transport regulator homolog 1 (E. coli)

A_44_P208688

2.58

Pof1b

302328

premature ovarian failure 1B

A_44_P370033

2.57

Ptpre

114767

protein tyrosine phosphatase, receptor type, E

A_44_P667206

2.57

Arl6ip1

293551

ADP-ribosylation factor-like 6 interacting protein 1

A_44_P473754

2.56

Son

304092

Son DNA binding protein

A_44_P116300

2.56

Ppargc1b

291567

peroxisome proliferator-activated receptor gamma,

coactivator 1 beta

A_44_P838798

2.55

Med1

497991

mediator complex subunit 1

A_42_P660129

2.55

Casp12

156117

caspase 12

A_44_P684579

2.52

Zbtb39

299510

zinc finger and BTB domain containing 39

A_44_P187830

2.51

Urb1

304104

URB1 ribosome biogenesis 1 homolog (S. cerevisiae)

A_44_P915229

2.50

Wdr89

314243

WD repeat domain 89

TABLE 5

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P336772

2.50

Josd1

315134

Josephin domain containing 1

A_44_P358216

2.50

Pank4

171053

pantothenate kinase 4

A_42_P597258

2.49

Ska2

287598

spindle and kinetochore associated complex subunit 2

A_44_P148161

2.49

Rrm1

685579

ribonucleotide reductase M1

A_43_P23126

2.48

Klf11

313994

Kruppel-like factor 11

A_44_P426823

2.45

Phc1

312690

polyhomeotic-like 1 (Drosophila)

A_44_P774203

2.45

Birc6

313876

baculoviral IAP repeat-containing 6

A_44_P283399

2.44

Cask

29647

calcium/calmodulin-dependent serine protein kinase (MAGUK

family)

A_43_P21324

2.44

Mesdc2

308796

mesoderm development candidate 2

A_44_P456968

2.43

St5

308944

suppression of tumorigenicity 5

A_43_P21033

2.43

Smyd5

312503

SET and MYND domain containing 5

A_44_P256534

2.43

Heca

308624

headcase homolog (Drosophila)

A_44_P148370

2.42

Plagl2

296281

pleiomorphic adenoma gene-like 2

A_42_P759427

2.42

Strn4

308392

striatin, calmodulin binding protein 4

A_44_P581181

2.42

Ftsjd1

292016

FtsJ methyltransferase domain containing 1

A_44_P729621

2.42

Maml1

303101

mastermind like 1 (Drosophila)

A_42_P684264

2.41

RGD1561817

294917

similar to Traf2 and NCK interacting kinase, splice variant 4

A_44_P525229

2.40

Anxa8

306283

annexin A8

A_44_P107423

2.40

Nup153

25281

nucleoporin 153

A_42_P532103

2.40

Anxa2

56611

annexin A2

A_44_P182061

2.40

Trappc2

501550

trafficking protein particle complex 2

A_44_P653272

2.40

Gpr155

311730

G protein-coupled receptor 155

A_42_P782490

2.39

Zfhx3

307829

zinc finger homeobox 3

A_44_P729898

2.38

Morc2

289736

MORC family CW-type zinc finger 2

A_44_P899288

2.38

Lrsam1

311866

leucine rich repeat and sterile alpha motif containing 1

A_44_P210198

2.38

Polg

85472

polymerase (DNA directed), gamma

A_44_P243534

2.35

RGD1311307

361238

similar to 1300014I06Rik protein

A_44_P182479

2.35

Kcnk4

116489

potassium channel, subfamily K, member 4

A_44_P397423

2.35

Tmcc3

314751

transmembrane and coiled-coil domain family 3

A_44_P381270

2.34

RGD1562136

501195

similar to D1Ertd622e protein

A_44_P258949

2.33

Pde4b

24626

phosphodiesterase 4B, cAMP specific

A_44_P302613

2.33

Lkap

170946

limkain b1

A_44_P548507

2.33

Chn1

84030

chimerin (chimaerin) 1

A_44_P853102

2.33

Dusp16

297682

dual specificity phosphatase 16

A_43_P21031

2.32

Pex5

312703

peroxisomal biogenesis factor 5

A_44_P191512

2.32

Ns5atp9

300795

NS5A (hepatitis C virus) transactivated protein 9

A_44_P344181

2.32

Map6

29457

microtubule-associated protein 6

A_44_P391057

2.32

Angptl4

362850

angiopoietin-like 4

A_44_P287958

2.32

Dnmt3b

444985

DNA (cytosine-5-)-methyltransferase 3 beta

A_44_P1032305

2.31

Fert2

301737

fer (fms/fps related) protein kinase, testis specific 2

A_43_P19499

2.31

Gzf1

311508

GDNF-inducible zinc finger protein 1

A_44_P340163

2.31

Plcb4

25031

phospholipase C, beta 4

A_44_P395928

2.31

Ap4m1

304344

adaptor-related protein complex 4, mu 1 subunit

TABLE 6

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P787936

2.31

Stat3

25125

signal transducer and activator of transcription 3

A_44_P1014018

2.31

Rkhd1

299613

ring finger (C3HC4 type) and KH domain containing 1

A_44_P652103

2.30

LOC691223

691223

hypothetical protein LOC691223

A_44_P457169

2.29

Dcp2

291604

DCP2 decapping enzyme homolog (S. cerevisiae)

A_44_P477322

2.29

Rtn4

83765

reticulon 4

A_44_P1021361

2.29

Tlr3

364594

toll-like receptor 3

A_44_P1022458

2.29

Tubb6

307351

tubulin, beta 6

A_44_P298210

2.29

Casp8

64044

caspase 8

A_44_P222799

2.28

Nin

299117

ninein (GSK3B interacting protein)

A_44_P149087

2.28

Fyn

25150

FYN oncogene related to SRC, FGR, YES

A_43_P17988

2.28

Tmem77

362011

transmembrane protein 77

A_44_P396115

2.28

Maml3

310405

mastermind like 3 (Drosophila)

A_42_P589201

2.27

Slc15a4

246280

solute carrier family 15, member 4

A_44_P170496

2.26

Sqrdl

691966

sulfide quinone reductase-like (yeast)

A_44_P558636

2.26

Ppp1r9b

84686

protein phosphatase 1, regulatory subunit 9B

A_44_P177448

2.26

Pafah1b1

83572

platelet-activating factor acetylhydrolase, isoform 1b, subunit 1

A_44_P885147

2.26

Def8

307973

differentially expressed in FDCP 8 homolog (mouse)

A_44_P428997

2.25

Fam164a

310244

family with sequence similarity 164, member A

A_44_P407894

2.25

Znf746

312303

zinc finger protein 746

A_42_P473389

2.25

Bmyc

311807

brain expressed myelocytomatosis oncogene

A_42_P680545

2.24

RGD1305235

292267

similar to RIKEN cDNA 1700052N19

A_42_P489223

2.23

Thada

313865

thyroid adenoma associated

A_44_P503022

2.23

Hip1

192154

huntingtin interacting protein 1

A_44_P228839

2.23

Pgrmc2

361940

progesterone receptor membrane component 2

A_44_P403621

2.22

Sgk3

171498

serum/glucocorticoid regulated kinase 3

A_44_P117148

2.22

Baz1a

314126

bromodomain adjacent to zinc finger domain, 1A

A_44_P607486

2.21

Foxn3

314374

forkhead box N3

A_44_P257206

2.21

Map3k12

25579

mitogen activated protein kinase kinase kinase 12

A_44_P618385

2.21

Efhd2

298609

EF-hand domain family, member D2

A_44_P191055

2.21

Pogz

310658

pogo transposable element with ZNF domain

A_44_P1030766

2.20

Pcf11

361605

PCF11, cleavage and polyadenylation factor subunit, homolog

(S. cerevisiae)

A_44_P112050

2.20

RGD1560523

498211

similar to S-adenosylmethionine synthetase gamma form

(Methionine adenosyltransferase)

A_44_P792035

2.19

Mcart1

313241

mitochondrial carrier triple repeat 1

A_44_P382226

2.19

Sox4

364712

SRY (sex determining region Y)-box 4

A_42_P484663

2.18

Calcoco1

246047

calcium binding and coiled coil domain 1

A_43_P15113

2.18

Myom1

316740

myomesin 1

A_42_P519018

2.17

Mtmr9

282584

myotubularin related protein 9

A_44_P210440

2.17

Akap13

293024

A kinase (PRKA) anchor protein 13

A_44_P1011464

2.17

Rhobtb1

309722

Rho-related BTB domain containing 1

A_44_P116443

2.17

Matr3

29150

matrin 3

A_44_P22150

2.16

LOC684297

684297

similar to PHD finger protein 8

A_44_P402675

2.16

Rxrb

361801

retinoid X receptor beta

A_44_P443542

2.15

Mars2

316403

methionyl-tRNA synthetase 2, mitochondrial

TABLE 7

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P391706

2.15

Abcb8

362302

ATP-binding cassette, sub-family B (MDR/TAP), member 8

A_43_P12905

2.15

Bik

114496

BCL2-interacting killer (apoptosis-inducing)

A_44_P652131

2.15

Med22

499762

mediator complex subunit 22

A_44_P531758

2.14

Cryab

25420

crystallin, alpha B

A_44_P357870

2.14

Cpt1a

25757

carnitine palmitoyltransferase 1a, liver

A_42_P669388

2.14

Cnksr1

298545

connector enhancer of kinase suppressor of Ras 1

A_44_P980916

2.14

Kirrel3

315546

kin of IRRE like 3 (Drosophila)

A_44_P698085

2.13

Arl13a

501617

ADP-ribosylation factor-like 13A

A_44_P622113

2.13

Sept8

303135

septin 8

A_44_P229079

2.12

Rb1

24708

retinoblastoma 1

A_44_P258497

2.12

Prmt2

499420

protein arginine methyltransferase 2

A_44_P422082

2.12

Ccnb3

317389

cyclin B3

A_44_P823666

2.11

Zhx2

314988

zinc fingers and homeoboxes 2

A_44_P991114

2.10

Pds5b

304218

PDS5, regulator of cohesion maintenance, homolog B

(S. cerevisiae)

A_44_P180060

2.09

Hdac6

84581

histone deacetylase 6

A_43_P13161

2.09

Prdm4

170820

PR domain containing 4

A_44_P256345

2.08

Smarcad1

312398

SWI/SNF-related, matrix-associated actin-dependent

regulator of chromatin, subfamily a, containing DEAD/H box 1

A_44_P398587

2.08

Usp9x

363445

ubiquitin specific peptidase 9, X-linked

A_43_P14872

2.07

Sdc1

25216

syndecan 1

A_44_P131290

2.07

Arhgap28

301709

Rho GTPase activating protein 28

A_44_P208464

2.07

Rad17

310034

RAD17 homolog (S. pombe)

A_44_P996369

2.06

Sorbs1

686098

sorbin and SH3 domain containing 1

A_43_P15281

2.06

Tp63

246334

tumor protein p63

A_44_P410455

2.06

Wdr48

363164

WD repeat domain 48

A_44_P148044

2.06

Hnrpll

313842

heterogeneous nuclear ribonucleoprotein L-like

A_44_P837227

2.06

LOC687994

687994

similar to 82-kD FMRP Interacting Protein

A_44_P536282

2.06

Sptbn1

305614

spectrin, beta, non-erythrocytic 1

A_44_P793692

2.05

Dtx3l

498089

deltex 3-like (Drosophila)

A_44_P494439

2.05

Cnnm2

294014

cyclin M2

A_44_P541053

2.04

Fbln5

29158

fibulin 5

A_42_P815727

2.04

Usp48

362636

ubiquitin specific protease 48

A_44_P1007347

2.03

Tgm2

56083

transglutaminase 2, C polypeptide

A_44_P304880

2.03

RGD1564776

366473

similar to ornithine decarboxylase-like protein

A_42_P678414

2.02

RGD1563106

303238

similar to novel protein

A_44_P191615

2.02

Taok1

286993

TAO kinase 1

A_44_P473524

2.02

Snx30

298033

sorting nexin family member 30

A_44_P147702

2.02

Utp6

360574

UTP6, small subunit (SSU) processome component, homolog

(yeast)

A_43_P23262

2.02

Cytip

311047

cytohesin 1 interacting protein

A_43_P12682

2.02

Rest

83618

RE1-silencing transcription factor

A_44_P279208

2.01

Eif4g3

298573

eukaryotic translation initiation factor 4 gamma, 3

A_44_P1032103

2.01

Adnp2

307236

ADNP homeobox 2

A_43_P22174

2.00

Wdr1

360950

WD repeat domain 1

A_44_P575034

2.00

Hiat1

100134827

hippocampus abundant gene transcript 1

TABLE 8

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P576954

2.00

Ddx58

297989

DEAD (Asp-Glu-Ala-Asp) box polypeptide 58

A_44_P140332

2.00

RGD1359600

307343

LEA 4 domain containing protein RGD1359600

A_43_P19525

2.00

Arhgap12

307016

Rho GTPase activating protein 12

A_44_P574253

1.99

Dapk2

300799

death-associated kinase 2

A_44_P824032

1.99

Nup160

311182

nucleoporin 160

A_44_P702258

1.99

Eea1

314764

early endosome antigen 1

A_43_P11363

1.98

LOC363188

363188

similar to ubiquitin protein ligase E3 component n-recognin 2

A_44_P763063

1.98

RGD1563437

291356

similar to KIAA1217

A_44_P638511

1.98

Paqr5

315741

progestin and adipoQ receptor family member V

A_44_P821966

1.97

LOC500684

500684

hypothetical protein LOC500684

A_44_P306840

1.96

Ddhd1

305816

DDHD domain containing 1

A_42_P806899

1.96

Kdm6b

363630

lysine (K)-specific demethylase 6B

A_44_P834393

1.96

Ube2d1

361831

ubiquitin-conjugating enzyme E2D 1, UBC4/5 homolog (yeast)

A_44_P229501

1.96

Rhoc

295342

ras homolog gene family, member C

A_44_P125042

1.96

LOC361100

361100

similar to topoisomerase (DNA) II beta

A_44_P116591

1.95

Cx3cl1

89808

chemokine (C-X3-C motif) ligand 1

A_44_P178561

1.95

LOC100302372

100302372

hypothetical protein LOC100302372

A_44_P335175

1.95

RGD1311892

289399

similar to hypothetical protein FLJ10901

A_44_P543917

1.95

Fbxo42

362646

F-box protein 42

A_42_P789482

1.95

Daam1

314212

dishevelled associated activator of morphogenesis 1

A_44_P123033

1.94

Klhl5

305351

kelch-like 5 (Drosophila)

A_44_P177845

1.94

Fbxo30

308283

F-box protein 30

A_44_P144188

1.94

Syt12

191595

synaptotagmin XII

A_42_P762924

1.94

Herc1

315771

hect (homologous to the E6-AP (UBE3A) carboxyl terminus)

domain and RCC1 (CHC1)-like domain (RLD) 1

A_44_P249161

1.93

Dab2ip

192126

DAB2 interacting protein

A_44_P466314

1.93

Tacc2

309025

transforming, acidic coiled-coil containing protein 2

A_44_P1045515

1.93

Sipa1l1

246212

signal-induced proliferation-associated 1 like 1

A_44_P267799

1.92

Ets2

304063

v-ets erythroblastosis virus E26 oncogene homolog 2 (avian)

A_44_P537673

1.92

Zbtb43

311872

zinc finger and BTB domain containing 43

A_44_P250620

1.92

Fhdc1

295161

FH2 domain containing 1

A_43_P12767

1.91

Pdlim5

64353

PDZ and LIM domain 5

A_44_P163189

1.90

Stxbp5

81022

syntaxin binding protein 5 (tomosyn)

A_44_P669345

1.90

Hdac8

363481

histone deacetylase 8

A_44_P450214

1.90

Sestd1

295678

SEC14 and spectrin domains 1

A_42_P597638

1.90

Acot2

192272

acyl-CoA thioesterase 2

A_44_P637827

1.89

Fam117b

363236

family with sequence similarity 117, member B

A_44_P935860

1.88

Nrip1

304157

nuclear receptor interacting protein 1

A_44_P265807

1.88

Aldh3a2

65183

aldehyde dehydrogenase 3 family, member A2

A_43_P10919

1.88

Exosc8

295050

exosome component 8

A_44_P508239

1.88

Shmt1

287379

serine hydroxymethyltransferase 1 (soluble)

A_44_P163816

1.88

Lrrc57

311346

leucine rich repeat containing 57

A_44_P539012

1.87

Rell2

361313

RELT-like 2

TABLE 9

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P699909

1.87

Zfyve16

499508

zinc finger, FYVE domain containing 16

A_42_P762829

1.87

Cebpd

25695

CCAAT/enhancer binding protein (C/EBP), delta

A_44_P484793

1.87

Tnrc6b

192178

trinucleotide repeat containing 6B

A_44_P363549

1.87

Nsun6

307148

NOL1/NOP2/Sun domain family, member 6

A_44_P744113

1.86

Dennd5a

308942

DENN/MADD domain containing 5A

A_44_P335377

1.86

Fbxw5

362081

F-box and WD repeat domain containing 5

A_42_P506402

1.86

Slc25a25

246771

solute carrier family 25 (mitochondrial carrier, phosphate

carrier), member 25

A_44_P539035

1.86

Dtwd1

296119

DTW domain containing 1

A_44_P992908

1.85

Hspb1

24471

heat shock protein 1

A_42_P640641

1.84

Homer2

29547

homer homolog 2 (Drosophila)

A_44_P387324

1.83

Nhej1

363251

nonhomologous end-joining factor 1

A_43_P10213

1.83

Etnk1

312828

ethanolamine kinase 1

A_44_P314312

1.83

Nol9

313744

nucleolar protein 9

A_42_P815566

1.82

Med23

309565

mediator complex subunit 23

A_42_P511015

1.82

Nup133

292085

nucleoporin 133

A_44_P562894

1.82

Rnd1

362993

Rho family GTPase 1

A_42_P621736

1.81

Rplp1

140661

ribosomal protein, large, P1

A_44_P576056

1.81

Ankfy1

303292

ankyrin repeat and FYVE domain containing 1

A_44_P520829

1.81

Pik3r4

363131

phosphoinositide-3-kinase, regulatory subunit 4

A_42_P586141

1.81

Plcd1

24655

phospholipase C, delta 1

A_44_P337351

1.81

Cxcl12

24772

chemokine (C-X-C motif) ligand 12 (stromal cell-derived

factor 1)

A_44_P929844

1.80

Lix1l

499677

Lix1 homolog (mouse)-like

A_44_P221056

1.80

Ralbp1

84014

ralA binding protein 1

A_44_P315562

1.80

Cald1

25687

caldesmon 1

A_44_P897591

1.79

LOC302495

302495

hypothetical LOC302495

A_44_P561538

1.79

Cdcp1

301082

CUB domain containing protein 1

A_44_P355758

1.79

Ralb

116546

v-ral simian leukemia viral oncogene homolog B (ras related;

GTP binding protein)

A_44_P477116

1.78

Pcyt1a

140544

phosphate cytidylyltransferase 1, choline, alpha

A_43_P20133

1.78

Skil

114208

SKI-like oncogene

A_44_P318466

1.78

Trim25

494338

tripartite motif-containing 25

A_44_P175875

1.78

Pigv

366478

phosphatidylinositol glycan anchor biosynthesis, class V

A_44_P404198

1.77

Mapk3

50689

mitogen activated protein kinase 3

A_43_P19455

1.77

Pard6g

307237

par-6 partitioning defective 6 homolog gamma (C. elegans)

A_44_P439473

1.77

Klf7

363243

Kruppel-like factor 7 (ubiquitous)

A_44_P224259

1.77

Pxn

360820

paxillin

A_44_P459918

1.76

Dnaja4

300721

DnaJ (Hsp40) homolog, subfamily A, member 4

A_44_P454872

1.76

Cnot1

291841

CCR4-NOT transcription complex, subunit 1

A_44_P313283

1.76

Capn2

29154

calpain 2

A_43_P12384

1.76

Birc3

78971

baculoviral IAP repeat-containing 3

A_44_P545945

1.76

Frmd8

309172

FERM domain containing 8

A_44_P358627

1.76

Chd2

308738

chromodomain helicase DNA binding protein 2

A_44_P850979

1.76

Prpf39

314171

PRP39 pre-mRNA processing factor 39 homolog (S. cerevisiae)

TABLE 10

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P173156

1.75

Taf5

294018

TAF5 RNA polymerase II, TATA box binding protein (TBP)-

associated factor

A_43_P19916

1.75

Eif4enif1

305468

eukaryotic translation initiation factor 4E nuclear import factor 1

A_43_P15633

1.75

Pkn2

207122

protein kinase N2

A_44_P639763

1.75

Fam179b

314169

family with sequence similarity 179, member B

A_44_P929706

1.75

Kdm2a

361700

lysine (K)-specific demethylase 2A

A_44_P402750

1.75

Gda

83585

guanine deaminase

A_44_P914528

1.75

Ccdc21

362622

coiled-coil domain containing 21

A_44_P151582

1.75

Il17re

362417

interleukin 17 receptor E

A_42_P526712

1.74

Eif4e3

297481

eukaryotic translation initiation factor 4E member 3

A_44_P920712

1.74

Dock8

499337

dedicator of cytokinesis 8

A_44_P397763

1.74

Nolc1

64896

nucleolar and coiled-body phosphoprotein 1

A_44_P372501

1.74

Sult1a1

83783

sulfotransferase family, cytosolic, 1A, phenol-preferring,

member 1

A_44_P516818

1.74

Ankrd17

289521

ankyrin repeat domain 17

A_44_P383360

1.73

Trim2

361970

tripartite motif-containing 2

A_44_P466111

1.73

Nfib

29227

nuclear factor I/B

A_44_P469584

1.73

Cd14

60350

CD14 molecule

A_43_P12519

1.73

S100a10

81778

S100 calcium binding protein A10

A_44_P699502

1.73

LOC687105

687105

hypothetical protein LOC687105

A_43_P23298

1.72

Ccdc123

292811

coiled-coil domain containing 123

A_44_P226478

1.72

RGD1309537

501203

similar to Myosin regulatory light chain 2-A, smooth muscle

isoform (Myosin RLC-A)

A_44_P591548

1.72

Pols

306672

polymerase (DNA directed) sigma

A_44_P472461

1.72

Aplf

500247

aprataxin and PNKP like factor

A_44_P117270

1.72

Gulp1

314543

GULP, engulfment adaptor PTB domain containing 1

A_44_P819401

1.72

Slc40a1

170840

solute carrier family 39 (iron-regulated transporter), member 1

A_43_P20415

1.72

Tfdp2

300947

Transcription factor Dp-2 (E2F dimerization partner 2)

A_44_P1055082

1.71

Dyrk2

314862

dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2

A_44_P472996

1.71

Anxa5

25673

annexin A5

A_44_P1003794

1.71

Nr3c1

24413

nuclear receptor subfamily 3, group C, member 1

A_42_P749968

1.71

RGD1306862

287596

similar to RIKEN cDNA 1200011M11

A_44_P827926

1.71

Rkhd2

307271

ring finger and KH domain containing 2

A_43_P11098

1.71

Cdc42bpb

113960

CDC42 binding protein kinase beta (DMPK-like)

A_44_P100507

1.71

Numa1

308870

nuclear mitotic apparatus protein 1

A_44_P335402

1.71

Mocos

361300

molybdenum cofactor sulfurase

A_44_P1040642

1.70

Mafb

54264

v-maf musculoaponeurotic fibrosarcoma oncogene homolog B

(avian)

A_44_P1033739

1.70

Akap11

498549

A kinase (PRKA) anchor protein 11

A_44_P100009

1.70

Diaph1

307483

diaphanous homolog 1 (Drosophila)

A_44_P711020

1.70

Znrf1

362367

zinc and ring finger 1

A_43_P14954

1.70

Sos1

313845

Son of sevenless homolog 1 (Drosophila)

A_43_P12245

1.70

Rhob

64373

ras homolog gene family, member B

A_44_P319349

1.70

Pkp2

287925

plakophilin 2

TABLE 11

ProbeName

RC

GeneSymbol

EntrezGene ID

GeneName

A_44_P143061

1.69

Srf

501099

serum response factor (c-fos serum response element-

binding transcription factor)

A_44_P1007449

1.69

RGD1310159

304500

similar to acetyl-coA dehydrogenase -related (111.6 kD)

(5G231)

A_44_P142242

1.69

Cdh1

83502

cadherin 1

A_44_P401284

1.69

Rnf19a

362900

ring finger protein 19A

A_42_P802550

1.69

Pdlim1

54133

PDZ and LIM domain 1

A_44_P105004

1.69

Csnk1g3

64823

casein kinase 1, gamma 3

A_44_P164007

1.68

Hectd1

362736

HECT domain containing 1

A_44_P338962

1.68

Taf5l

307927

TAF5-like RNA polymerase II, p300/CBP-associated factor

(PCAF)-associated factor

A_43_P19059

1.68

Noc3l

361753

nucleolar complex associated 3 homolog (S. cerevisiae)

A_44_P927671

1.68

Nfat5

307820

nuclear factor of activated T-cells 5

A_44_P482953

1.68

RGD1562529

311372

similar to hypothetical protein FLJ21439

A_44_P1033595

1.68

Atl3

309187

atlastin GTPase 3

A_44_P244138

1.68

Ptpn9

266611

protein tyrosine phosphatase, non-receptor type 9

A_44_P1034793

1.67

Ncdd4l

291553

neural precursor cell expressed, developmentally down-

regulated 4-like

A_44_P345159

1.67

Wipi1

303630

WD repeat domain, phosphoinositide interacting 1

A_44_P591729

1.67

Gnal

24611

guanine nucleotide binding protein, alpha stimulating, olfactory

type

A_44_P246114

1.67

Tle1

362533

transducin-like enhancer of split 1 (E(sp1) homolog,

Drosophila)

A_44_P998638

1.67

Pprc1

294007

peroxisome proliferator-activated receptor gamma,

coactivator-related 1

A_43_P12708

1.67

Src

83805

v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog

(avian)

A_44_P465931

1.67

Clic1

406864

chloride intracellular channel 1

A_43_P11255

1.67

Secisbp2l

296115

SECIS binding protein 2-like

A_44_P1019407

1.67

Rnf103

84508

ring finger protein 103

A_43_P21480

1.67

Zfp772

308320

zinc finger protein 772

A_44_P956272

1.67

Ogt

26295

O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-

acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase)

A_44_P653818

1.67

Zcchc2

304695

zinc finger, CCHC domain containing 2

A_44_P393064

1.66

Clasp1

304740

cytoplasmic linker associated protein 1

A_44_P398969

1.66

Gtpbp2

363195

GTP binding protein 2

A_42_P538617

1.66

Ift172

116475

intraflagellar transport 172 homolog (Chlamydomonas)

A_44_P494768

1.66

LOC500625

500625

Hypothetical protein LOC500625

A_42_P639047

1.66

RGD1309350

293613

similar to transthyretin (4L369)

A_44_P1037041

1.66

Map3k6

313022

mitogen-activated protein kinase kinase kinase 6

A_44_P153774

1.66

Plce1

114633

phospholipase C, epsilon 1

A_44_P229776

1.65

Nedd4

25489

neural precursor cell expressed, developmentally down-

regulated 4

A_44_P1022889

1.65

Cpne3

313087

copine III

A_42_P524707

1.65

Prim2

301323

primase, DNA, polypeptide 2