• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.89-89
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• 2003

본 연구에서는 1.7kb 및 3.1kb bovine $\beta$-casein promoter의 유전자 발현 조절능력을 알아보기 위해 1 7kb 및 3.1kb bovine $\beta$-casein promoter에 human Type II Collagen 유전자를 연결해서 DNA microinjection으로 형질전환생쥐를 생산하였다. 총 8마리의 founder생쥐(1.7kb collagen : 5마리, 3.1kb collagen 3마리)를 생산하였고 이 founder생쥐와 wild type 생쥐를 mating시켜서 $F_1 및 F_2$ 새끼를 얻었다. $F_1 및 F_2$새끼들에서 human Type II collagen 유전자의 transmission rate는 약 50%로 Mendel의 법칙에 따라 분리되어 안정적으로 유전자가 염색체에 정착되어 있음을 확인하였다. 이들 $F_1 및 F_2$새끼 중 암컷들을 임신시켜 분만 후 5-10 일경에 유선조직을 포함하여 여러 조직으로부터 RNA를 추출하여 Northern blotting 및 RT-PCR 방법을 이용하여 Type II collagen mRNA의 발현을 분석하였다. 유선에서의 발현은 1 7 kb 및 3.1 kb line별로 각각 1 line씩 발현되지 않았고, 그 외 line에서는 모두 발현되는 것으로 확인되었다. 유선에서의 Type II collagen mRNA 발형양은 1.7 kb 및 3.1 kb bovine $\beta$-casein promoter사이에서는 큰 차이를 나타내지 않았으나 1.7 kb promoter 형질전환생쥐의 경우 유선 이외 조직에서도 발현되는 양상을 나타내었고, 3.1kb promoter line에서는 유선특이적으로 발현시키는 양상을 나타내었다. 그러므로 bovine $\beta$-casein promoter의 1.7 kb와 3.1 kb 사이에 유선특이적 발현을 유도하는 조절부위가 있을 것으로 추정된다.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.421-427
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• 2012

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ${\beta}$-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ${\beta}$-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ${\beta}$-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ${\beta}$. In addition, the first intron of the porcine ${\beta}$-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ${\beta}$, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ${\beta}$-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ${\beta}$-casein gene. The ${\beta}$-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ${\beta}$-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ${\beta}$-casein gene activity.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.71-71
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• 2002

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. ${\beta}$-casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of ${\beta}$-casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. ${\beta}$-casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of ${\beta}$-casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine ${\beta}$-casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.

• Development and Reproduction
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• v.13 no.3
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-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 $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ 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 $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.89-89
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• 2003

• 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

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.375-383
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• 1994

The human growth hormone (hGH) gene uder the control of the rat $\beta$-casein promoter gene was designed to produce transgenic mouse expressed hGH gene in only mammary gland. One hundred seventy two eggs microinjected were transferred to the oviducts of pseudopregnants and 43 offspring were delivered. By Southern blotting hybridization, 3 were transgenic with rat $\beta$-casein/hGH gene. The copy numbers of three transgenic founder were 1, 5, and 15, respectively. A radioimmunoassay was developed to quantitate the amount of expression of the hGH gene in mammary gland of transgenic mice. The amount of hGH was 13.3ng/ml in the lactating milk of one transgenic line, showing predominantly higher than 3.0ng/ml in milk of control mice. Therefore, our findings suggested that $\beta$-casein promoter may induce the tissue specific expression of structural 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.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.11
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• pp.1628-1633
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• 2001

A goat ${\beta}$-casein gene was cloned and sequenced. Our previous study had determined the nucleotide sequences of the 5' flanking region and the structural gene including all 9 exons. In the present study, investigations were done on the regulatory sequences in the 5' flanking region of the goat ${\beta}$-casein gene by aligning and comparing it with the same gene from other mammals. The results showed that -200/-1 bp of the 5' flanking sequences contained six conserved clusters, in which the sites of gene expression regulated by the transcription factor and hormone might exist. It showed that fourteen glucocorticoid receptor elements, two cAMP responsive elements, two SV40 virus enhancer core sequences, two OCT-1 binding elements and one CTF/NF-1 binding element were dispersed in the 5' flanking region of goat ${\beta}$-casein gene. Our findings are perhaps valuable for the elucidation of the molecular mechanisms that control the expression of the goat ${\beta}$-casein gene.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.43-43
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• 2003

This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (tPA) gene. Two different tPA genes containing bovine $\beta$-casein promoter and mouse uroplakin promoter were prepared for microinjection and confirmed the expression level of tPA protein from the CHO (Chinese hamster ovary) cell lines by gene transfection. Concentration of tPA expression from the six cell lines (all of CHO cells) were average 212.4 ng/ml. Reconstructed DNA to used the CHO cell were microinjected into the pronuclei of in vivo embryos The total of 2,307 zygotes were collected from 95 donors and 1,851 embryos were in 1-cell stage which were visualized the pronuclei for DNA microinjection. The concentration of linear DNA was 2.0 ng per microliter and injected into zygotes with two pronuclei on an inverted Nikon microscope equipped with narishige micromanipulator and modulation contrast optics. The 541 embryos injected with bovine $\beta$-casein promoter-tPA were transferred to 22 recipients. The 1,154 embryos injected with mouse uroplakin promoter-tPA were transferred to 51 recipients. Sixty nine offspring from 9 delivered sows were produced. We analysed the transgenes with PCR methods from 69 offsprings, but could not detect the PCR product from piglet tails DNA.