Skip to main content
SpringerLink
Log in

A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice

  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

A β-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3′ region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3′ end, with BLG sequences of exon 6/intron 6/exon 7 and the 3′ region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3′ flanking sequences. The BLG 5′ regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Archibald AL, McClenaghan M, Hornsey V, Simons JP and Clark AJ (1990) High-level expression of a foreign gene in a line of transgenic mice is modulated by chromosomal position effect. Mol Cell Biol 10: 1192–1198.

    Google Scholar 

  • Auffray C and Rougeon F (1980) Purification of mouse immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem 107: 303–314.

    Google Scholar 

  • Baolin L, Murphy KL, Laucirica R, Kittrell F, Medina D and Rosen JM (1998) A transgenic mouse model for mammary carcinogenesis. Oncogene 16: 997–1007.

    Google Scholar 

  • Barcellos-Hoff MH, Aggeler J, Ram TG and Bissell MJ (1989) Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105: 223–235.

    Google Scholar 

  • Barash I, Faerman A, Baruch A, Nathan M, Hurwitz DR and Shani M (1993) Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice. Transgenic Res 2: 266–276.

    Google Scholar 

  • Barash I, Faerman A, Ratovitsky T, Puzis R, Nathan M, Hurwitz DR and Shani M (1994) Ectopic expression of β-lactoglobulin/Human serum albumin fusion genes in transgenic mice: hormonal regulation and in situ localization. Transgenic Res 3: 141–151.

    Google Scholar 

  • Barash I, Nathan M, Keri R, Ilan N, ShaniMand Hurwitz DR (1996) Elements within the β-lactoglobulin gene inhibit expression of human serum albumin cDNA or minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice. Nucl Acids Res 24: 602–610.

    Google Scholar 

  • Barash, I, Faerman A, Richenstein M, Kari R, Damari G-M, Shani Mand Bissell MJ (1999) in vivo and in vitro expression of human serum albumin genomic sequences in the mammary epithelial cells with β-lactoglobulin and whey acidic protein promoters. Mol Rep Dev 52: 241–252.

    Google Scholar 

  • Barash I (1999) Prolactin and insulin synergize to regulate the translation modulator PHAS-I via mitogen-activated protein kinaseindependent but wortmannin-and rapamycin-sensitive pathway. Mol Cell Endocrinol 155: 37–49.

    Google Scholar 

  • Baruch A, Shani M and Barash I (1998) Insulin and prolactin synergize to induce translation of human serum albumin in the mammary gland of transgenic mice. Transgenic Res 7: 15–127.

    Google Scholar 

  • Bortner DM and Rosenberg MP (1995) Overexpression of cyclin A in the mammary gland of transgenic mice results in the induction of nuclear abnormalities and increased apoptosis. Cell Growth Diff 6: 1579–1589.

    Google Scholar 

  • Brinster RL, Allen JM, Behringer RR, Gelinas RE and Palmiter RD (1988) Intron increases transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 85: 836–840.

    Google Scholar 

  • Clark AJ, Harold G and Yull FE (1997) Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice. Nucl Acids Res 25: 1009–1014.

    Google Scholar 

  • Clarke RA, Sokol D, Rigby N, Ward K, Murray JD and Mackinlay AG (1994) Mammary gland-specific expression of bovine αS1-casein-derived transgenes in mice. Transgenics 1: 313–319.

    Google Scholar 

  • Colman A (1996) Production of proteins in the milk of transgenic livestock: problems, solutions and successes. Am J Clin Nutr 63: 639S–645S.

    Google Scholar 

  • Dale TC, Krnacik MJ, Schmidhauser C, Yang CL-Q, Bissell MJ and Rosen J (1992) High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3′ untranslated region.Mol Cell Biol 12: 905–914.

    Google Scholar 

  • Demmer J, Burdon TG, Djiane J, Watson CJ and Clark AJ (1995) The proximal milk protein binding factor binding site is required for the prolactin responsiveness of the sheep β-lactoglobulin promoter in Chinese hamster ovary cells. Mol Cell Endocrinol 107: 113–121.

    Google Scholar 

  • Devinoy E, Thepot D, Stinnakre MG, Fontaine ML, Grabowski H, Puissant C, Pavirani A and Houdebine LM (1994) High level of production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene target transgene expression to the mammary gland. Transgenic Res 3: 79–89.

    Google Scholar 

  • Edelman GM and Jones FS (1997) Gene regulation of cell adhesion molecules in neural morphogenesis. Acta Paediatr Suppl 422: 12–19.

    Google Scholar 

  • Franklein GC, Donovan M, Adam GIR, Holmgren L, Pfeifer-Ohlsson S and Ohlsson R (1991) Expression of the human PDGF-B gene is regulated by both positive and negative acting cell type-specific regulatory elements located in the first intron. EMBO J 10: 1365–1373.

    Google Scholar 

  • Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H and Hennighausen L (1994) Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 91: 9302–9306.

    Google Scholar 

  • Gross DS and Garrard WT (1988) Nuclease hypersensitive site in chromatin. Annu Rev Biochem 57: 159–197.

    Google Scholar 

  • Hurwitz DR, Nathan M, Barash I, Ilan N and Shani M (1994) Specific combinations of human serum albumin introns direct high levels of expression of albumin in transfected COS cells and in the milk of transgenic mice. Transgenic Res 3: 365–375.

    Google Scholar 

  • Kang YK, Lee CS, Chung AS and Lee KK (1998) Prolactininducible activity of the first intron of the bovine beta-casein gene. Mol Cell 30: 259–265.

    Google Scholar 

  • Lily C, Chao AJ, Kim SJ, Huang L, and Martinson HG (1999) Assembly of the cleavage and polyadenylation apparatus requires about 10 seconds in vivo and is faster for strong than for weak Poly(A) sites. Mol Cell Biol 19: 5588–5600.

    Google Scholar 

  • Liu K, Sandgren EP, Palmiter RD and Stein A (1995) Rat growth hormone gene introns stimulate nucleosome alignment in vitro and in transgenic mice. Proc Natl Acad Sci USA 92: 7724–7728.

    Google Scholar 

  • McKnight RA, Shamay A, Sankaran L, Wall RJ and Hennighausen L (1992) Matrix attachment regions can impart position independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci USA 89: 6943–6947.

    Google Scholar 

  • Myers CA, Schmidhauser C, Mellentin-Michelotti J, Fragoso G, Roskelley CD, Casperson G, Mossi R, Pujuguet P, Hager G and Bissell MJ (1998) Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation. Mol Cell Biol 18: 2184–2195.

    Google Scholar 

  • Ninomiya T, Hirabayashi M, Sagara J and Yuki A (1994) Functions of milk protein gene 5′ flanking regions on human growth hormone gene. Mol Reprod Dev 37: 276–283.

    Google Scholar 

  • Palmiter RD, Sandgren EP, Avabrock MR, Allen DD and Brinster RL (1991) Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci USA 88: 478–482.

    Google Scholar 

  • Pena RN, Folch JM, Sanchez A and Whitelaw CBA (1998) Chromatin structures of goat and sheep β-lactoglobulin gene differ. Biochem Biophys Res Comm 252: 649–653.

    Google Scholar 

  • Pierre S, Devinoy JE, Theron MC, Malienou-N'Gassa R, Puissant C and Houdebine LM(1992) A distal region enhances the prolactin induced promoter activity of the rabbit α-S1 casein gene. Mol Cell Endocrinol 87: 7–156.

    Google Scholar 

  • Proudfoot N (1991) Poly(A) signals. Cell 64: 671–674.

    Google Scholar 

  • Rotwein P, Bichell DP and Kikuchi K (1993) Multifunctional regulation of IGF-I gene expression. Mol Rep Dev 35: 358–364.

    Google Scholar 

  • Schmidhauser C, Bissell MJ, Myers CA, and Casperson J (1990) Extracellular matrix and hormones transcriptionally regulate bovine β-casein 5–sequences in stably transfected mouse mammary cells. Proc Natl Acad Sci USA 87: 9118–9122.

    Google Scholar 

  • Shani M, Barash I, Nathan M, Ricca G, Searfoss GH, Dekel I, Faerman A, Givol D and Hurwitz DR (1992) Expression of human serum albumin in the milk of transgenic mice. Transgenic Res 1: 195–208.

    Google Scholar 

  • van der Hoff MJB, Labruyere WT, Moorman AFM and Lamers WH (1993) Mammary gene expression is improved by use of a longer SV40 early polyadenylation cassette. Nucl Acids Res 21: 4987–4988.

    Google Scholar 

  • Wei Y, Yarus S, Greenberg NM, Whitsett J and Rosen JM (1995) Production of human surfactant protein C in milk of transgenic mice. Transgenic Res 4: 232–240.

    Google Scholar 

  • Watson CJ, Gordon KE, Robertson M and Clark AJ (1991) Interaction of DNA-binding proteins with a milk protein gene promoter in vitro: identification of a mammary gland-specific factor. Nucl Acids Res 19: 6603–6610.

    Google Scholar 

  • Webster J, Wallace RM, Clark AJ and Whitelaw CBA (1995) Tissue-specific, temporally regulated expression mediated by the proximal ovine β-lactoglobulin promoter in transgenic mice. Cell Mol Biol Res 41: 11–15.

    Google Scholar 

  • Webster J, Donofrio, G, Wallace R, Clark AJ and Whitelaw CB (1997) Intronic sequences modulate the sensitivity of betalactoglobulin transgene to position effect. Gene 193: 239–243.

    Google Scholar 

  • Whitelaw CBA, Archibald AL, Harris S, McClenaghan M, Simons PJ and Clark AJ (1991) Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice. Transgenic Res 1: 1–13.

    Google Scholar 

  • Whitelaw CBA, Harris S, McClenaghan M, Simons JP and Clark AJ (1992) Position-independent expression of the ovine β-lactoglobulin gene in transgenic mice. Biochem J 286: 31–39.

    Google Scholar 

  • Wright G, Carver A, Cottom D, Reevers D, Scott A, Simons P, Wilmut I, Carner I and Colman A (1991) High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. Bio/Technology 9: 830–834.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel

    Moshe Reichenstein, Helen Gottlieb, Golda-Meir Damari, Elena Iavnilovitch & Itamar Barash

Authors
  1. Moshe Reichenstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Helen Gottlieb
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Golda-Meir Damari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elena Iavnilovitch
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Itamar Barash
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Reichenstein, M., Gottlieb, H., Damari, GM. et al. A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice. Transgenic Res 10, 445–456 (2001). https://doi.org/10.1023/A:1012064922126

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1012064922126

Search

Navigation