Characterization of Double Transgenic Mice Harboring Both Goat β-casein/hGH and Goat β-casein/hG-CSF Hybrid Genes

다국어 초록 (Multilingual Abstract)

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat β-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to 12.4 ㎎/㎖ for hGH and from 0.04 to 0.13 ㎎/㎖ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat β-casein promoter. Therefore goat β-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

참고문헌 (Reference)

1 Chen S, "Use of polymerase chain reaction for screening transgenic mice. In: White BA (ed.). Methods in Molecular Biology, V115, PCR protcols: Current Methods and Applications" Human Press, Totoma 74-80, 1993

2 Goldman IL, "Transgenic animals in medicine: integration and expression of foreign genes, theoretical and applied aspects" 10 : 274-285, 2004

3 Houdebine LM, "Transgenic animal bioreactors" 9 : 305-320, 2005

4 Yull F, "Transgene rescue in the mammary gland is associated with transcription but does not require translation of BLG transgenes" 6 : 11-17, 1997

5 Clark AJ, "The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins" 3 : 337-350, 1998

6 Chomczynski R, "Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction" 162 : 156-159, 1987

7 Clark AJ, "Rescuing transgene expression by co-integration" 10 : 1450-1454, 1992

8 Langley B, "Rescue of an MMTV transgene by co- integration reveals novel locus control properties of the ovine β-lactoglobulin gene that confer locus commitment to heterogeneous tissues" 7 : 205-212, 1998

9 Echelard Y, "Recombinant protein production in transgenic animals" 7 : 537-540, 1996

10 Drews R, "Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin" 92 : 10462-10466, 1995

11 Demain AL, "Production of recombinant proteins by microbes and higher organisms" 27 : 297-306, 2009

12 Gordon K, "Production of human tissue plasminogen activator in transgenic mouse milk" 5 : 1183-1187, 1987

13 Ko JH, "Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat" 9 : 215-222, 2000

14 Wilmut I, "Modification of milk composition" 41 : I35-I46, 1990

15 Clark AJ, "Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice" 25 : 1009-1014, 1997

16 이철상, "Integration and Expression of Goat β-Casein/hGH Hybrid Gene in a Transgenic Goat" 한국동물번식학회 30 (30): 293-299, 2006

17 Prunkard D, "High-level expression of recombinant human fibrinogen in the milk of transgenic mice" 14 : 867-871, 1996

18 이철상, "Goat β-casein Promoter Directs hGH Expression at a High Level and in a Mammary-Specific Manner in Transgenic Mice" 한국유전학회 28 (28): 131-137, 2006

19 Palmiter RD, "Germ-line transformation of mice" 20 : 465-499, 1986

20 Reddy VB, "Expression of human growth hormone in the milk of transgenic mice" 2 : 15-29, 1991

21 McKnight RA, "Expression of genomic and cDNA transgenes after co-integration in transgenic mice" 4 : 39-43, 1995

22 John DCA, "Expression of an engineered form of recombinant procollagen in mouse milk" 17 : 385-389, 1999

23 Bae CS, "Enhanced secretion of human granulocyte colony- stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration" 57 : 600-609, 1998

24 Hunter CV, "Developments in transgenic technology: applications for medicine" 11 : 293-298, 2005

25 Mire-Sluis AR, "Cytokines: from technology to therapeutics" 17 : 319-325, 1999

26 Barash I, "Co-integration of beta- lactoglobulin/human serum albumin hybrid genes with the entire beta-lactoglobulin gene or the matrix attachment region element: repression of human serum albumin and beta-lactoglobulin expression in the mammary gland and oglo regulation of the transgenes" 45 : 421-430, 1996

27 Lu YF, "Cloning of human G-CSF genomic gene and its expression in transgenic mice mammary gland" 26 : 281-287, 1999

28 Gabrilove JL, "Clinical applications of granulocyte colony stimulating factor" 6 : 187-191, 1992

29 Oh KB, "A hybrid bovine β-casein/bGH gene directs transgene expression to the lung and mammary gland of transgenic mice" 8 : 307-311, 1999