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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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Methylation of CMV promoter.(A) Level of CMV methylation increased in founder transgenic pigs from newborn to maturity. The increases in CMV methylation level from 26% to 40% (p = 1.000) and from 19% to 38% (p = 0.799) were observed in K25-2 and K25-3, respectively; (B) Level of CMV methylation increased in transgenic fibroblast cells over time in culture. The increase in CMV methylation level from 20 to 90 days was more than 3-fold from 26% to 81% (p = 0.053); (C) Variegation of CMV methylation in various tissues of transgenic pig. Hypermethylation and hypomethylation levels of CMV methylation were found in different tissues, but the difference was not significant (p = 0.153); (D) Level of CMV methylation in offspring transgenic pigs. The increase in CMV methylation level from 40% to 58% on average of offspring was detected (p = 0.537). Error bars denote standard deviations.
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pone-0006679-g005: Methylation of CMV promoter.(A) Level of CMV methylation increased in founder transgenic pigs from newborn to maturity. The increases in CMV methylation level from 26% to 40% (p = 1.000) and from 19% to 38% (p = 0.799) were observed in K25-2 and K25-3, respectively; (B) Level of CMV methylation increased in transgenic fibroblast cells over time in culture. The increase in CMV methylation level from 20 to 90 days was more than 3-fold from 26% to 81% (p = 0.053); (C) Variegation of CMV methylation in various tissues of transgenic pig. Hypermethylation and hypomethylation levels of CMV methylation were found in different tissues, but the difference was not significant (p = 0.153); (D) Level of CMV methylation in offspring transgenic pigs. The increase in CMV methylation level from 40% to 58% on average of offspring was detected (p = 0.537). Error bars denote standard deviations.

Mentions: We detected CMV methylation levels in ears of newborn and mature transgenic pigs, and observed an increase from 26% to 40% (p = 1.000) and from 19% to 38% (p = 0.799) in K25-2 and K25-3, respectively (Fig. 5A).


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)

Methylation of CMV promoter.(A) Level of CMV methylation increased in founder transgenic pigs from newborn to maturity. The increases in CMV methylation level from 26% to 40% (p = 1.000) and from 19% to 38% (p = 0.799) were observed in K25-2 and K25-3, respectively; (B) Level of CMV methylation increased in transgenic fibroblast cells over time in culture. The increase in CMV methylation level from 20 to 90 days was more than 3-fold from 26% to 81% (p = 0.053); (C) Variegation of CMV methylation in various tissues of transgenic pig. Hypermethylation and hypomethylation levels of CMV methylation were found in different tissues, but the difference was not significant (p = 0.153); (D) Level of CMV methylation in offspring transgenic pigs. The increase in CMV methylation level from 40% to 58% on average of offspring was detected (p = 0.537). Error bars denote standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006679-g005: Methylation of CMV promoter.(A) Level of CMV methylation increased in founder transgenic pigs from newborn to maturity. The increases in CMV methylation level from 26% to 40% (p = 1.000) and from 19% to 38% (p = 0.799) were observed in K25-2 and K25-3, respectively; (B) Level of CMV methylation increased in transgenic fibroblast cells over time in culture. The increase in CMV methylation level from 20 to 90 days was more than 3-fold from 26% to 81% (p = 0.053); (C) Variegation of CMV methylation in various tissues of transgenic pig. Hypermethylation and hypomethylation levels of CMV methylation were found in different tissues, but the difference was not significant (p = 0.153); (D) Level of CMV methylation in offspring transgenic pigs. The increase in CMV methylation level from 40% to 58% on average of offspring was detected (p = 0.537). Error bars denote standard deviations.
Mentions: We detected CMV methylation levels in ears of newborn and mature transgenic pigs, and observed an increase from 26% to 40% (p = 1.000) and from 19% to 38% (p = 0.799) in K25-2 and K25-3, respectively (Fig. 5A).

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