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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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Copy number of GFP.(A) Absolute quantitative real-time PCR analysis of GFP copy number from newborn to maturity in founder transgenic pigs. There was no statistically significant decline in K25-2 (p = 0.099), but the decline in K25-3 was significant (p = 0.016); (B) Southern blots analysis of GFP copy number in newborn and mature transgenic pigs; (C) Absolute quantitative real-time PCR analysis of GFP copy number in transgenic fibroblast cells. Copy number of GFP declined in cells over time in culture. The decline was significant (p<0.001); (D) Variegation of GFP copy number in various tissues of transgenic pig. Variegation of GFP copies was shown in different tissues (p = 0.059); (E) Absolute quantitative real-time PCR analysis of GFP copy number in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (F) Southern blots analysis of GFP copy number in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.
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pone-0006679-g003: Copy number of GFP.(A) Absolute quantitative real-time PCR analysis of GFP copy number from newborn to maturity in founder transgenic pigs. There was no statistically significant decline in K25-2 (p = 0.099), but the decline in K25-3 was significant (p = 0.016); (B) Southern blots analysis of GFP copy number in newborn and mature transgenic pigs; (C) Absolute quantitative real-time PCR analysis of GFP copy number in transgenic fibroblast cells. Copy number of GFP declined in cells over time in culture. The decline was significant (p<0.001); (D) Variegation of GFP copy number in various tissues of transgenic pig. Variegation of GFP copies was shown in different tissues (p = 0.059); (E) Absolute quantitative real-time PCR analysis of GFP copy number in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (F) Southern blots analysis of GFP copy number in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.

Mentions: In order to determine the correlation of transgene expression with copy number, we examined the GFP copy number in ears of newborn and mature transgenic pigs. A decline in copy number was found by absolute quantitative real-time PCR (Fig. 3A) and Southern blot analysis (Fig. 3B). Although a significant decline was not detected in K25-2 (p = 0.099), the decline in K25-3 was significant (p = 0.016). A more than 4-copy drop was observed in K25-2, and a more than 6-copy drop was observed in K25-3 (table 1). Transgenic animals show a difference in transgene copy number [27], [28], but a decline in the copy number with aging is not a common observation.


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)

Copy number of GFP.(A) Absolute quantitative real-time PCR analysis of GFP copy number from newborn to maturity in founder transgenic pigs. There was no statistically significant decline in K25-2 (p = 0.099), but the decline in K25-3 was significant (p = 0.016); (B) Southern blots analysis of GFP copy number in newborn and mature transgenic pigs; (C) Absolute quantitative real-time PCR analysis of GFP copy number in transgenic fibroblast cells. Copy number of GFP declined in cells over time in culture. The decline was significant (p<0.001); (D) Variegation of GFP copy number in various tissues of transgenic pig. Variegation of GFP copies was shown in different tissues (p = 0.059); (E) Absolute quantitative real-time PCR analysis of GFP copy number in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (F) Southern blots analysis of GFP copy number in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2723931&req=5

pone-0006679-g003: Copy number of GFP.(A) Absolute quantitative real-time PCR analysis of GFP copy number from newborn to maturity in founder transgenic pigs. There was no statistically significant decline in K25-2 (p = 0.099), but the decline in K25-3 was significant (p = 0.016); (B) Southern blots analysis of GFP copy number in newborn and mature transgenic pigs; (C) Absolute quantitative real-time PCR analysis of GFP copy number in transgenic fibroblast cells. Copy number of GFP declined in cells over time in culture. The decline was significant (p<0.001); (D) Variegation of GFP copy number in various tissues of transgenic pig. Variegation of GFP copies was shown in different tissues (p = 0.059); (E) Absolute quantitative real-time PCR analysis of GFP copy number in offspring transgenic pigs. The decline from founder to offspring was significant (p<0.001); (F) Southern blots analysis of GFP copy number in offspring transgenic pigs. Error bars denote standard deviations. Neg., non-transgenic pig.
Mentions: In order to determine the correlation of transgene expression with copy number, we examined the GFP copy number in ears of newborn and mature transgenic pigs. A decline in copy number was found by absolute quantitative real-time PCR (Fig. 3A) and Southern blot analysis (Fig. 3B). Although a significant decline was not detected in K25-2 (p = 0.099), the decline in K25-3 was significant (p = 0.016). A more than 4-copy drop was observed in K25-2, and a more than 6-copy drop was observed in K25-3 (table 1). Transgenic animals show a difference in transgene copy number [27], [28], but a decline in the copy number with aging is not a common observation.

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