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Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs.

Kong Q, Wu M, Huan Y, Zhang L, Liu H, Bou G, Luo Y, Mu Y, Liu Z - PLoS ONE (2009)

Bottom Line: Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases.However, inheritance and expression instability of the transgene in transgenic animals is a major limitation.Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression.

View Article: PubMed Central - PubMed

Affiliation: College of life science, Northeast Agricultural University of China, Harbin, People's Republic of China.

ABSTRACT
Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.

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Expression of GFP.(A) Relative real-time RT-PCR analysis of GFP mRNA expression from newborn to maturity in founder transgenic pigs. The decline was significant in both K25-2 and K25-3 (p<0.001); (B) Western blots analysis of GFP protein from newborn to maturity in founder transgenic pigs; (C) Relative real-time RT-PCR analysis of GFP mRNA expression in transgenic fibroblast cells. The decline of mRNA from 20 to 90 days was significant (p<0.001); (D) Flow cytometry analysis of the percentage of phenotype positive cells. The decline of percentage of positive cells from 20 to 90 days was also significant (p<0.001); (E) Relative real-time RT-PCR analysis of GFP mRNA expression in various tissues of the transgenic pig. Variegation of GFP mRNA expression was shown in different tissues (p<0.001); (F) Western blots analysis of GFP protein in various tissues; (G) Relative real-time RT-PCR analysis of GFP mRNA expression in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (H) Western blots analysis of GFP protein in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.
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pone-0006679-g001: Expression of GFP.(A) Relative real-time RT-PCR analysis of GFP mRNA expression from newborn to maturity in founder transgenic pigs. The decline was significant in both K25-2 and K25-3 (p<0.001); (B) Western blots analysis of GFP protein from newborn to maturity in founder transgenic pigs; (C) Relative real-time RT-PCR analysis of GFP mRNA expression in transgenic fibroblast cells. The decline of mRNA from 20 to 90 days was significant (p<0.001); (D) Flow cytometry analysis of the percentage of phenotype positive cells. The decline of percentage of positive cells from 20 to 90 days was also significant (p<0.001); (E) Relative real-time RT-PCR analysis of GFP mRNA expression in various tissues of the transgenic pig. Variegation of GFP mRNA expression was shown in different tissues (p<0.001); (F) Western blots analysis of GFP protein in various tissues; (G) Relative real-time RT-PCR analysis of GFP mRNA expression in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (H) Western blots analysis of GFP protein in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.

Mentions: We analyzed the change in GFP expression in relation to aging in ear tissues of transgenic pigs. A significant decline in the mRNA level was observed from newborn to maturity in both K25-2 and K25-3 (p<0.001). The mRNA level decreased about 1-fold and 3-fold in K25-2 and K25-3, respectively (Fig. 1A). These results were consistent with results from Western blot analysis (Fig. 1B). These data indicate that the transgene expression level decreases with aging in vivo.


Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs.

Kong Q, Wu M, Huan Y, Zhang L, Liu H, Bou G, Luo Y, Mu Y, Liu Z - PLoS ONE (2009)

Expression of GFP.(A) Relative real-time RT-PCR analysis of GFP mRNA expression from newborn to maturity in founder transgenic pigs. The decline was significant in both K25-2 and K25-3 (p<0.001); (B) Western blots analysis of GFP protein from newborn to maturity in founder transgenic pigs; (C) Relative real-time RT-PCR analysis of GFP mRNA expression in transgenic fibroblast cells. The decline of mRNA from 20 to 90 days was significant (p<0.001); (D) Flow cytometry analysis of the percentage of phenotype positive cells. The decline of percentage of positive cells from 20 to 90 days was also significant (p<0.001); (E) Relative real-time RT-PCR analysis of GFP mRNA expression in various tissues of the transgenic pig. Variegation of GFP mRNA expression was shown in different tissues (p<0.001); (F) Western blots analysis of GFP protein in various tissues; (G) Relative real-time RT-PCR analysis of GFP mRNA expression in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (H) Western blots analysis of GFP protein in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2723931&req=5

pone-0006679-g001: Expression of GFP.(A) Relative real-time RT-PCR analysis of GFP mRNA expression from newborn to maturity in founder transgenic pigs. The decline was significant in both K25-2 and K25-3 (p<0.001); (B) Western blots analysis of GFP protein from newborn to maturity in founder transgenic pigs; (C) Relative real-time RT-PCR analysis of GFP mRNA expression in transgenic fibroblast cells. The decline of mRNA from 20 to 90 days was significant (p<0.001); (D) Flow cytometry analysis of the percentage of phenotype positive cells. The decline of percentage of positive cells from 20 to 90 days was also significant (p<0.001); (E) Relative real-time RT-PCR analysis of GFP mRNA expression in various tissues of the transgenic pig. Variegation of GFP mRNA expression was shown in different tissues (p<0.001); (F) Western blots analysis of GFP protein in various tissues; (G) Relative real-time RT-PCR analysis of GFP mRNA expression in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (H) Western blots analysis of GFP protein in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.
Mentions: We analyzed the change in GFP expression in relation to aging in ear tissues of transgenic pigs. A significant decline in the mRNA level was observed from newborn to maturity in both K25-2 and K25-3 (p<0.001). The mRNA level decreased about 1-fold and 3-fold in K25-2 and K25-3, respectively (Fig. 1A). These results were consistent with results from Western blot analysis (Fig. 1B). These data indicate that the transgene expression level decreases with aging in vivo.

Bottom Line: Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases.However, inheritance and expression instability of the transgene in transgenic animals is a major limitation.Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression.

View Article: PubMed Central - PubMed

Affiliation: College of life science, Northeast Agricultural University of China, Harbin, People's Republic of China.

ABSTRACT
Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.

Show MeSH
Related in: MedlinePlus