Quantitative analysis of lentiviral transgene expression in mice over seven generations.
Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken together, these data suggested that transgenic lines with long term stable expression and no position effect can be established by lentiviral transgenesis.
Wang
Yong
Y
Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
Song
Yong-tao
YT
Liu
Qin
Q
Liu
Cang'e
C
Wang
Lu-lu
LL
Liu
Yu
Y
Zhou
Xiao-yang
XY
Wu
Jun
J
Wei
Hong
H
D000818:Animals; D018899:CpG Islands / Q000235:genetics; D003433:Crosses, Genetic; D019175:DNA Methylation; D005260:Female; D005470:Fluorometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D013047:Specific Pathogen-Free Organisms; D013997:Time Factors; D019076:Transgenes; D025822:Ubiquitin C / Q000235:genetics; D016662:Virus Integration
D011993:Recombinant Fusion Proteins; D025822:Ubiquitin C; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins