Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

[1]  Wei Sun,et al.  The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct. , 2012, Journal of biotechnology.

[2]  Yong Cheng,et al.  Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure. , 2012, Theriogenology.

[3]  Yong Cheng,et al.  Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary‐specific expression of human lactoferrin in transgenic mice , 2012, Molecular reproduction and development.

[4]  Meng Li,et al.  Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin. , 2012, Theriogenology.

[5]  Xiaorong Zhang,et al.  In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos , 2012, Biotechnology Letters.

[6]  Fuyin Xiong,et al.  The High-Level Expression of Human Tissue Plasminogen Activator in the Milk of Transgenic Mice with Hybrid Gene Locus Strategy , 2012, Molecular Biotechnology.

[7]  H. Niemann,et al.  Advances in farm animal transgenesis. , 2011, Preventive veterinary medicine.

[8]  Wang Yongsheng,et al.  Human lysozyme gene (hLYZ) transgenic bovine embryos produced by somatic cell nuclear transfer. , 2011 .

[9]  L. An,et al.  Cloned kids derived from caprine mammary gland epithelial cells. , 2009, Theriogenology.

[10]  Yong Cheng,et al.  [Production of cloned goats by transfer of nuclei from transfected caprine mammary gland epithelial cells]. , 2009, Sheng wu gong cheng xue bao = Chinese journal of biotechnology.

[11]  Yanrong Zhou,et al.  A mWAP–hLF hybrid gene locus gave extremely high level expression of human lactoferrin in the milk of transgenic mice , 2009, Transgenic Research.

[12]  G. Gong,et al.  Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows. , 2008, Journal of dairy science.

[13]  Y. Liu,et al.  Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin , 2008, PloS one.

[14]  J. Robl,et al.  Commercialising genetically engineered animal biomedical products. , 2008, Reproduction, fertility, and development.

[15]  P. Fox,et al.  Compositional, microbiological, biochemical, volatile profile and sensory characterization of four Italian semi-hard goats' cheeses , 2007, Journal of Dairy Research.

[16]  Liu Si-guo,et al.  Effect of cytoplast on the development of inter‐subspecies nuclear transfer reconstructed goat embryo , 2007, Molecular reproduction and development.

[17]  H. Matsui,et al.  Bovine α-Lactalbumin Stimulates Mucus Metabolism in Gastric Mucosa , 2007 .

[18]  C. Lin,et al.  Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows , 2005, Cell biology international.

[19]  I. Barash,et al.  BLG‐e1 – A novel regulatory element in the distal region of the β‐lactoglobulin gene promoter , 2005, FEBS letters.

[20]  J. Kim,et al.  Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. , 2005, Theriogenology.

[21]  M. Shani,et al.  Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice , 1994, Transgenic Research.

[22]  Ning Li,et al.  Variable Expression of Human Lactoferrin Gene in Mice Milk Driven by Its 90 KB Upstream Flanking Sequences , 2004, Animal biotechnology.

[23]  I. Barash,et al.  A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice , 2001, Transgenic Research.

[24]  L. Houdebine.  Transgenic animal bioreactors , 2004, Transgenic Research.

[25]  B. Whitelaw,et al.  A future for transgenic livestock , 2003, Nature Reviews Genetics.

[26]  S. Stice,et al.  In Vitro Development of Bovine Nuclear Transfer Embryos from Transgenic Clonal Lines of Adult and Fetal Fibroblast Cells of the Same Genotype1 , 2002, Biology of reproduction.

[27]  W. Hurley,et al.  Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk. , 2002, Journal of animal science.

[28]  H. Green,et al.  Cloned Transgenic Offspring Resulting from Somatic Cell Nuclear Transfer in the Goat: Oocytes Derived from Both Follicle-Stimulating Hormone-Stimulated and Nonstimulated Abattoir-Derived Ovaries1 , 2001, Biology of reproduction.

[29]  R. Prather,et al.  Clonal Lines of Transgenic Fibroblast Cells Derived from the Same Fetus Result in Different Development When Used for Nuclear Transfer in Pigs1 , 2001, Biology of reproduction.

[30]  M. Wheeler,et al.  Short communication: effects of increased expression of alpha-lactalbumin in transgenic mice on milk yield and pup growth. , 2001, Journal of dairy science.

[31]  L. Schibler,et al.  Position-independent and copy-number-related expression of a goat bacterial artificial chromosome alpha-lactalbumin gene in transgenic mice. , 1999, The Biochemical journal.

[32]  M. Bissell,et al.  In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β‐lactoglobulin and whey acidic protein promoters , 1999, Molecular reproduction and development.

[33]  M. Wheeler,et al.  Production of bovine alpha-lactalbumin in the milk of transgenic pigs. , 1998, Journal of animal science.

[34]  D. Stuart,et al.  Crystal structure of human α-lactalbumin at 1·7 Å resolution , 1991 .