• Reproductive and Developmental Biology
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• v.41 no.3
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• pp.51-55
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• 2017

The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of ${\beta}$-casein gene and expressed using the gene regulatory DNA sequence of bovine ${\beta}$-casein gene. The knock-in vector consists of 5' arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3' arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5' terminal of bIGF-1 gene and inserted into exon 7 of the ${\beta}$-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the ${\beta}$-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine ${\beta}$-casein gene.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.31-36
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• 1991

Yeast expression plasmids containing an appropriate leader sequence and bovine $\beta$-casein cDNA were constructed to produce $\beta$-casein for the study of its functional characteristics. Two kinds of expression systems for $\beta$-casein were constructed using pCGYl444 as a precursor plasmid. This plasmid is a yeast-E. coli shuttle vector which contains the chelatin promoter. The plasmid pISB202 contains the invertase leader sequence and $\beta$-casein gene. The plasmid pDEB303 contains the original bovine $\beta$-casein leader sequence gene. These two plasmids were introduced into S. cerevisiae AB116 which is a strain deficient in the major yeast proteinases. Each clone was grown in minimal media for 24 h before induction by $CuSO_4$. The cells were thus grown under expression conditions. Both strains harbouring pISB202 and pDEB303 expressed bovine $\beta$-casein. The $\beta$-casein was detected using immunochemical staining after western blot. Secretion of $\beta$-casein was detected in the culture broth. The estimated amount of secreted $\beta$-casein was approximately 50 ${\MU}g$/l.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.237-244
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• 1998

To investigate the fidelity of transgene transmission and expression, we produced transgenic mice carrying bovine $\beta$-casein/bovine grwoth hormone(bGH) fusion gene and examined transmission efficiency and expression level of the transgene in the founders and their progeny. The transgene was composed of 1.8 kb bovine $\beta$-casein promoter and 2.1 kb bGH gene. Ten transgenic mice were produced. Milk and mammary gland were collected from eight transgenic lines at 10-day lactation and a, pp.ied to Western and Northern blot analyses. The bGH expression was detected in four of them. The concentrations of bGH in milk were highly variable from 4$\mu\textrm{g}$/ml to 600$\mu\textrm{g}$/ml depending on the lines. The bGH mRNA level in mammary gland was closely correlated with the bGH concentration in milk in each transgenic line. These results indicated that bGH transgene expression was a, pp.opriately regulated in the mammary gland and secreted into milk in transgenic mice. By using two transgenic lines(#2, #7) secreting a considerable amoung of bGH into their milk, the inferitance and maintenance of transgenic phenotype were assessed in successive four generations. The mean transmission frequencies of transgene in lines #2 and #7 were 34% and 40%, respectively. The bGH concentration in milk were 80, 240, 120, 60$\mu\textrm{g}$/ml in each G0(generation 0), G2, G3, G4 generation of line #2 and 600, 1600, 860, 900$\mu\textrm{g}$/ml in each G1. G2, G3, G4 generation of line #7. These results demonstrated that bovine $\beta$-casein/bGH gene was stably transmitted from generation to generation in a Menelian fashion in trasgenic mice and consistenly expressed in their milk throughout the generations, although there was a little variation in the transmission frequency and expression level of the transgene between generations.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.4
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• pp.541-547
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• 1993

A phage clone containing the partial ${\alpha}_{S1}$-casein gene was isolated from a bovine genomic library by using mixed probes of ovine ${\alpha}_{S1}$-, ${\beta}$- and ${\kappa}$-casein cDNAs. Restriction enzyme mapping analysis for 14.6 kb revealed that the map was in conflict with the report of Meade et al. (1990), especially in the 3'-end fragment. Sequence analysis of 12.6 kb revealed a high AT/GC ratio (1.64); we have identified eight exon sequences according to the bovine ${\alpha}_{S1}$-casein cDNA sequence. The same exon/intron splice junction sequence was observed between these exons. We suggest that the bovine ${\alpha}_{S1}$-casein gene night contain a minimum of 18 exons and the full length is approximately 18-19 kb.

• BMB Reports
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• v.31 no.4
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• pp.413-417
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• 1998

Transgenic mice containing a bovine ${\beta}-Casein/Human$ lysozyme fusion gene (pBZ) were generated in order to produce human lysozyme in their milk. The expression vector was a quadripartite fusion consisting of a 2 kb upstream DNA of the bovine ${\beta}-casein$ gene, human lysozyme gene, intron II of the rabbit ${\beta}-globin$ gene, and the polyadenylation/termination signals of SV40 DNA. Fertilized mouse zygotes were microinjected with pBZ, then transferred into the oviduct of foster mothers. Out of 20 mice born, 11 survived until postweaning and three were identified as positivetransgenic by Southern blot analysis (one male and two females). The founder mice were mated to BCFl mice to produce transgenic progeny. It was confirmed by RT-PCR and Northern blot analyses that the transgene was specifically expressed in the mammary gland of the founder mice. Furthermore, the artificial introns within the transgenic RNA was proven to be correctly spliced out as judged by RT-PCR analysis. These results indicated that transgenic mice generated in this study properly expressed the human lysozyme RNA in their mammary gland.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.6
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• pp.806-813
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• 2010

Animal bioreactors have been regarded as alternative tools for the production of limited human therapeutic proteins. The mammary glands of cattle are optimal tissues to produce therapeutic proteins that cannot be produced in large amounts in traditional systems based on microorganisms and eukaryotic cells. In this study, two knock-in vectors, pBCTPOKI-6 and pBCTPOKI-10, which target the hTPO gene on the bovine beta-casein locus, were designed to develop cloned transgenic cattle. The pBCTPOKI-6 and pBCTPOKI-10 vectors expressed hTPO protein in culture medium at a concentration of 774 pg/ml and 1,867 pg/ml, respectively. Successfully, two targeted cell clones were obtained from the bovine fibroblasts transfected with the pBCTPOKI-6 vector. Cloned embryos reconstructed with the targeted nuclei showed a lower in vitro developmental competence than those with the wild-type nuclei. After transfer of the cloned embryos into recipients, 7 pregnancies were detected at 40 to 60 days of gestation, but failed to develop to term. The results are the first trial for targeting of a human gene on the bovine milk protein gene locus, providing the potential for a large-scale production of therapeutic proteins in the animal bioreactor system.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.10
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• pp.1397-1401
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• 2003

The aim of this study was to determine genotypes of four genetic markers and to investigate their association with milk production traits in Brown Swiss cattle imported to Slovakia. The bovine $\kappa$-casein, $\beta$-lactoglobulin, growth hormone and prolactin genotypes of 107 cows were identified by polymerase chain reaction. Effects all four genetic markers on milk, fat, protein and lactose yields and fat, protein and lactose percentage were estimated from a data set of 249 lactations. The frequency of desirable B allele of $\kappa$-casein gene to milk production was 0.46, alleles A of $\beta$-lactoglobulin gene was 0.55, allele and L of growth hormone gene was 0.45 and allele A and B of bovine prolactin gene were 0.61 and 0.39. The results of milk production obtained in our work showed that BB genotypes of $\kappa$-CN gene, AA genotypes of $\beta$-LG gene, LL genotypes of bGH gene were significantly associated with better milk production traits, mainly about the fat content. Association of a bovine prolactin genotypes with milk production were not found.

• BMB Reports
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• v.28 no.1
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• pp.57-61
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• 1995

The expression of a recombinant human lactoferrin is reported in mouse HC11 mammary epithelial cells. Expression of human lactoferrin (hLF) was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To improve the hLF expression level in a cell culture system, two artificial introns were also introduced to construct expression vectors. One intron was a hybrid-splice signal consisting of bovine ${\beta}$-casein intron 1 and rabbit ${\beta}$-globin intron II. The other intron was a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. The hybrid intron moderately elevated hLF expression, whereas intron 8 alone did not express any detectable amount of hLF as judged by Northem and Western blot analyses. When the two introns were used together they contributed to a synergistic elevation of hLF expression. These data indicate that artificial introns on both sides of the hLF cDNA were necessary to increase expression of cDNA.

• Korean Journal of Agricultural Science
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• v.28 no.2
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• pp.92-98
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• 2001

Lactofenicin is an antibacterial peptide fragment (about 5 kD) derived from lactoferrin (80 kD) that displays the various biological functions. The production of a human lactoferricin (Lactoferricin H) in mouse HC11 mammary epithelial cells was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To express lactoferricin H in this cell culture system, constructed a hybride-splice signal consisting of bovine ${\beta}$-casein intron I and rabbit ${\beta}$-globin intron II, and a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. Expression of lactofenicin H from this expression vector was identified by RT-PCR, northern and dot blot analysis. RT-PCR using total RNA of HC11 cells transfected with pBL1-cin expression vector yielded a product identified as having a size of the 150bp. Northern blot analysis was identified about 2.3 kb. In dot blot analysis, recombinant lactofenicin H was recognized with anti-human lactofrrnin polyclonal antibody.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.45-45
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• 2004