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

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.

Figure 1. 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.

Figure 2. Variegation of GFP expression in various tissues of the transgenic pig.

A to L: different tissues, namely uterus, spleen, ovary, muscle, liver, intestine, lung, tongue, kidney, stomach, heart and adipose, were visualized by HE staining. A1 to L1: tissues under normal light. A2 to L2: tissues under UV light.

Figure 3. 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.

Figure 4. Bisulfite sequence data for a representative region of CMV promoter.

Native P is the native sequence of the CMV promoter. The sequences from clones obtained after bisulfite treatment of DNA samples were aligned to the native sequence. For example, in control P cytosine residues were not methylated and therefore converted to thymidine by the bisulfite treatment. CpG and non-CpG cytosines were highlighted on a red and pink background, respectively.

Figure 5. 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.