• 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.

• 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.

• Korean Journal of Agricultural Science
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• v.25 no.2
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• pp.216-224
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• 1998

This study was performed to offer the basic and applicable data for improvement of Korean cattle and dairy cattle, according to finding the genetic construction obtained from analysis of genetic polymorphisms of ${\beta}$-lactoglobulin and ${\kappa}$-casein loci related Korean cattle and Holstein cows using PCR-RFLP. Genomic DNA used in this study was prepared from the blood of 253 individuals of Korean cattle in Korean Native Cattle Improvement Center, NLCF, and the blood of 113 individuals of Holstein cows in National Livestock Research Institute. The results obtained are summarized as follows : 1. This study confirmed amplified products of 530bp and 262bp fragments obtained from the amplification of ${\beta}$-lactoglobulin and ${\kappa}$-casein loci in Korean cattle and Holstein breed by PCR. 2. The ${\beta}$-lactoglobulin AA genotype showed 153bp and 109bp fragments, and ${\beta}$-lactoglobulin AB genotype showed 153bp, 109bp, 79bp and 74bp fragments, and BB genotype showed 109bp, 79bp and 74bp fragments in amplified products of ${\beta}$-lactoglobulin loci with the restricted enzyme digestion of Hae III. 3. The ${\kappa}$-casein AA genotype showed a 530bp fragment, and ${\kappa}$-casein AB genotype showed 530bp, 344bp and 186bp fragments, and BB genotype showed 344bp and 186bp fragments in amplified products of ${\kappa}$-casein loci with the restricted enzyme digestion of Taq I. 4. On ${\beta}$-lactoglobulin genotypes and gene frequencies, Korean cattle were 6.72%, 26.09% and 67.19% for AA, AB and BB genotypes, and ${\beta}$-lactoglobulin A and B alleles were 0.197 and 0.803, and Holstein were 35.40%, 56.64% and 7.96% for AA, AB and BB genotypes, and ${\beta}$-lactoglobulin A and B alleles were 0.637 and 0.363, respectively. 5. On ${\kappa}$-casein genotypes and gene frequencies, Korean cattle were 46.25%, 39.13% and 14.62% for AA, AB and BB genotypes, and ${\kappa}$-casein A and B alleles were 0.658 and 0.342, and Holstein were 60.18% and 38.94% and 0.88% for AA, AB and BB genotypes, and ${\kappa}$-casein A and B alleles were 0.796 and 0.204, respectively. 6. As a consequence, the gene frequency was 0.197 and 0.803 for ${\beta}$-lactoglobulin A and B alleles, and 0.658 and 0.342 for ${\kappa}$-casein A and B alleles in Korea cattle, but was 0.637 and 0.363 for ${\beta}$-lactoglobulin A and B alleles, and 0.796 and 0.204 for ${\kappa}$-casein A and B alleles in Holstein, respectively.

• 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.

• 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

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.6
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• pp.743-749
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• 2004

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

• 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.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.293-299
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• 2006

In order to generate transgenic goats expressing human growth hormone (hGH) in their mammary glands, goat ${\beta}-Casein/hGH$ hybrid gene was introduced into goat zygotes by pronuclear microinjection. DNA-injected embryos were transferred to the oviduct of recipients at 2-cell stage or to the uterus at morula/blastocyst stage after cultivation in glutathione-supplemented mSOF medium in vitro. Pregnancy and survival rate were not significantly different between 2-cell embryos and morula/blastocysts transferred to oviduct and uterus, respectively. One transgenic female goat was generated from 153 embryos survived from DNA injection. Southern blot analysis revealed that the transgenic goat harbored single-copy transgene with a partial deletion in its sequences. Despite of the partial sequence deletion, the transgene was successfully expressed hGH at the level of $72.1{\pm}15.1{\mu}g/ml$ in milk throughout lactation period, suggesting that the sequence deletion had occurred in non-essential part of the transgene for the transgene expression. Unfortunately, however, the transgene was not transmitted to her offspring during three successive breeding seasons. These results demonstrated that goat ${\beta}-casein/hGH$ gene was integrated into the transgenic goat genome in a mosaic fashion with a partial sequence deletion, which could result in a low level expression of hGH and a failure of transgene transmission.

• 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.

• Reproductive and Developmental Biology
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• v.41 no.1
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• pp.7-16
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• 2017

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the ${\beta}-casein$ gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5' arm region and 1.8 kb or 0.64 kb of 3' arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5' terminal of endostatin gene and inserted into exon 7 of the ${\beta}-casein$ gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3' arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine ${\beta}-casein$ gene in the mammary gland.