Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Nov 1;311(Pt 3):929–937. doi: 10.1042/bj3110929

Expression analysis of the individual bovine beta-, alpha s2- and kappa-casein genes in transgenic mice.

M Rijnkels 1, P M Kooiman 1, P J Krimpenfort 1, H A de Boer 1, F R Pieper 1
PMCID: PMC1136091  PMID: 7487953

Abstract

To identify cis-acting regulatory elements involved in the regulation of expression of the casein genes, the bovine beta-, alpha s2- and kappa-casein genes were isolated from cosmid libraries and introduced into the murine germline. Bovine casein expression was analysed at the RNA and protein level. The bovine beta-casein gene, including 16 kb of 5'- and 8 kb of 3'-flanking region, appeared to be expressed in all 12 transgenic mouse lines analysed. In 50% of these lines expression levels in milk exceeded 1 mg/ml. Three lines displayed expression levels comparable with or well above (20 mg/ml) the beta-casein levels in bovine milk. Transgene expression was restricted to the mammary gland. Strong induction of expression occurred at parturition and thus resembled the bovine rather than the murine pattern. In spite of this high-level tissue-specific and developmentally regulated expression, beta-casein expression levels were integration-site-dependent, suggesting that not all elements involved in regulation of expression were included in this beta-casein clone. Neither the bovine alpha s2- nor the kappa-casein gene, including 8 kb and 5 kb of 5'- and 1.5 kb and 19 kb of 3'-flanking sequences respectively, were properly expressed in transgenic mice. However, they were transcribed in stably transfected mouse mammary epithelial cells. This indicates that regulatory elements required for high-level, mammary gland-specific expression are not present in the alpha s2- and kappa-casein clones used in this study and are probably located elsewhere in the casein gene locus.

Full text

PDF
929

Images in this article

931Figure 2
on p.931
932Figure 3
on p.932
932Figure 4
on p.932
933Figure 5
on p.933
934Figure 6
on p.934
934Figure 7
on p.934
935Figure 8
on p.935

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander L. J., Stewart A. F., Mackinlay A. G., Kapelinskaya T. V., Tkach T. M., Gorodetsky S. I. Isolation and characterization of the bovine kappa-casein gene. Eur J Biochem. 1988 Dec 15;178(2):395–401. doi: 10.1111/j.1432-1033.1988.tb14463.x. [DOI] [PubMed] [Google Scholar]
  2. Altiok S., Groner B. beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter. Mol Cell Biol. 1994 Sep;14(9):6004–6012. doi: 10.1128/mcb.14.9.6004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  4. Ball R. K., Friis R. R., Schoenenberger C. A., Doppler W., Groner B. Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J. 1988 Jul;7(7):2089–2095. doi: 10.1002/j.1460-2075.1988.tb03048.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bühler T. A., Bruyère T., Went D. F., Stranzinger G., Bürki K. Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits. Biotechnology (N Y) 1990 Feb;8(2):140–143. doi: 10.1038/nbt0290-140. [DOI] [PubMed] [Google Scholar]
  6. Dillon N., Grosveld F. Transcriptional regulation of multigene loci: multilevel control. Trends Genet. 1993 Apr;9(4):134–137. doi: 10.1016/0168-9525(93)90208-y. [DOI] [PubMed] [Google Scholar]
  7. Doppler W., Groner B., Ball R. K. Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line. Proc Natl Acad Sci U S A. 1989 Jan;86(1):104–108. doi: 10.1073/pnas.86.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doppler W., Höck W., Hofer P., Groner B., Ball R. K. Prolactin and glucocorticoid hormones control transcription of the beta-casein gene by kinetically distinct mechanisms. Mol Endocrinol. 1990 Jun;4(6):912–919. doi: 10.1210/mend-4-6-912. [DOI] [PubMed] [Google Scholar]
  9. Eisenstein R. S., Rosen J. M. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level. Mol Cell Biol. 1988 Aug;8(8):3183–3190. doi: 10.1128/mcb.8.8.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gaensler K. M., Kitamura M., Kan Y. W. Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11381–11385. doi: 10.1073/pnas.90.23.11381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodman R. E., Schanbacher F. L. Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland. Biochem Biophys Res Commun. 1991 Oct 15;180(1):75–84. doi: 10.1016/s0006-291x(05)81257-4. [DOI] [PubMed] [Google Scholar]
  12. Groenen M. A., Dijkhof R. J., Verstege A. J., van der Poel J. J. The complete sequence of the gene encoding bovine alpha s2-casein. Gene. 1993 Jan 30;123(2):187–193. doi: 10.1016/0378-1119(93)90123-k. [DOI] [PubMed] [Google Scholar]
  13. Groenen M. A., Dijkhof R. J., van der Poel J. J., van Diggelen R., Verstege E. Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene. Nucleic Acids Res. 1992 Aug 25;20(16):4311–4318. doi: 10.1093/nar/20.16.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grosveld F., van Assendelft G. B., Greaves D. R., Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987 Dec 24;51(6):975–985. doi: 10.1016/0092-8674(87)90584-8. [DOI] [PubMed] [Google Scholar]
  15. Gupta P., Rosen J. M., D'Eustachio P., Ruddle F. H. Localization of the casein gene family to a single mouse chromosome. J Cell Biol. 1982 Apr;93(1):199–204. doi: 10.1083/jcb.93.1.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hayes H., Petit E., Bouniol C., Popescu P. Localization of the alpha-S2-casein gene (CASAS2) to the homoeologous cattle, sheep, and goat chromosomes 4 by in situ hybridization. Cytogenet Cell Genet. 1993;64(3-4):281–285. doi: 10.1159/000133593. [DOI] [PubMed] [Google Scholar]
  17. Heilig R., Lemaire C., Mandel J. L. A 230kb cosmid walk in the Duchenne muscular dystrophy gene: detection of a conserved sequence and of a possible deletion prone region. Nucleic Acids Res. 1987 Nov 25;15(22):9129–9142. doi: 10.1093/nar/15.22.9129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hennighausen L. G., Sippel A. E. Characterization and cloning of the mRNAs specific for the lactating mouse mammary gland. Eur J Biochem. 1982 Jun 15;125(1):131–141. doi: 10.1111/j.1432-1033.1982.tb06660.x. [DOI] [PubMed] [Google Scholar]
  19. Hennighausen L., Westphal C., Sankaran L., Pittius C. W. Regulation of expression of genes for milk proteins. Biotechnology. 1991;16:65–74. [PubMed] [Google Scholar]
  20. Lee C. S., Oka T. A pregnancy-specific mammary nuclear factor involved in the repression of the mouse beta-casein gene transcription by progesterone. J Biol Chem. 1992 Mar 25;267(9):5797–5801. [PubMed] [Google Scholar]
  21. Lee C. S., Oka T. Progesterone regulation of a pregnancy-specific transcription repressor to beta-casein gene promoter in mouse mammary gland. Endocrinology. 1992 Nov;131(5):2257–2262. doi: 10.1210/endo.131.5.1425425. [DOI] [PubMed] [Google Scholar]
  22. Lee K. F., Atiee S. H., Rosen J. M. Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice. Mol Cell Biol. 1989 Feb;9(2):560–565. doi: 10.1128/mcb.9.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee K. F., DeMayo F. J., Atiee S. H., Rosen J. M. Tissue-specific expression of the rat beta-casein gene in transgenic mice. Nucleic Acids Res. 1988 Feb 11;16(3):1027–1041. doi: 10.1093/nar/16.3.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maga E. A., Anderson G. B., Huang M. C., Murray J. D. Expression of human lysozyme mRNA in the mammary gland of transgenic mice. Transgenic Res. 1994 Jan;3(1):36–42. doi: 10.1007/BF01976025. [DOI] [PubMed] [Google Scholar]
  25. Meade H., Gates L., Lacy E., Lonberg N. Bovine alpha S1-casein gene sequences direct high level expression of active human urokinase in mouse milk. Biotechnology (N Y) 1990 May;8(5):443–446. doi: 10.1038/nbt0590-443. [DOI] [PubMed] [Google Scholar]
  26. Meier V. S., Groner B. The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction. Mol Cell Biol. 1994 Jan;14(1):128–137. doi: 10.1128/mcb.14.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ninomiya T., Hirabayashi M., Sagara J., Yuki A. Functions of milk protein gene 5' flanking regions on human growth hormone gene. Mol Reprod Dev. 1994 Mar;37(3):276–283. doi: 10.1002/mrd.1080370306. [DOI] [PubMed] [Google Scholar]
  28. Persuy M. A., Stinnakre M. G., Printz C., Mahe M. F., Mercier J. C. High expression of the caprine beta-casein gene in transgenic mice. Eur J Biochem. 1992 May 1;205(3):887–893. doi: 10.1111/j.1432-1033.1992.tb16854.x. [DOI] [PubMed] [Google Scholar]
  29. Pieper F. R., de Wit I. C., Pronk A. C., Kooiman P. M., Strijker R., Krimpenfort P. J., Nuyens J. H., de Boer H. A. Efficient generation of functional transgenes by homologous recombination in murine zygotes. Nucleic Acids Res. 1992 Mar 25;20(6):1259–1264. doi: 10.1093/nar/20.6.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roberts B., DiTullio P., Vitale J., Hehir K., Gordon K. Cloning of the goat beta-casein-encoding gene and expression in transgenic mice. Gene. 1992 Nov 16;121(2):255–262. doi: 10.1016/0378-1119(92)90129-d. [DOI] [PubMed] [Google Scholar]
  31. Rosen J. M., Bayna E., Lee K. F. Analysis of milk protein gene expression in transgenic mice. Mol Biol Med. 1989 Dec;6(6):501–509. [PubMed] [Google Scholar]
  32. Schedl A., Montoliu L., Kelsey G., Schütz G. A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature. 1993 Mar 18;362(6417):258–261. doi: 10.1038/362258a0. [DOI] [PubMed] [Google Scholar]
  33. Schmidhauser C., Bissell M. J., Myers C. A., Casperson G. F. Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9118–9122. doi: 10.1073/pnas.87.23.9118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schmidhauser C., Casperson G. F., Myers C. A., Sanzo K. T., Bolten S., Bissell M. J. A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression. Mol Biol Cell. 1992 Jun;3(6):699–709. doi: 10.1091/mbc.3.6.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmitt-Ney M., Doppler W., Ball R. K., Groner B. Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol. 1991 Jul;11(7):3745–3755. doi: 10.1128/mcb.11.7.3745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schmitt-Ney M., Happ B., Hofer P., Hynes N. E., Groner B. Mammary gland-specific nuclear factor activity is positively regulated by lactogenic hormones and negatively by milk stasis. Mol Endocrinol. 1992 Dec;6(12):1988–1997. doi: 10.1210/mend.6.12.1491685. [DOI] [PubMed] [Google Scholar]
  37. Thompson M. D., Dave J. R., Nakhasi H. L. Molecular cloning of mouse mammary gland kappa-casein: comparison with rat kappa-casein and rat and human gamma-fibrinogen. DNA. 1985 Aug;4(4):263–271. doi: 10.1089/dna.1985.4.263. [DOI] [PubMed] [Google Scholar]
  38. Threadgill D. W., Womack J. E. Genomic analysis of the major bovine milk protein genes. Nucleic Acids Res. 1990 Dec 11;18(23):6935–6942. doi: 10.1093/nar/18.23.6935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yu-Lee L. Y., Richter-Mann L., Couch C. H., Stewart A. F., Mackinlay A. G., Rosen J. M. Evolution of the casein multigene family: conserved sequences in the 5' flanking and exon regions. Nucleic Acids Res. 1986 Feb 25;14(4):1883–1902. doi: 10.1093/nar/14.4.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES