Abstract
Integration target site is the most important factor in successful production of transgenic animals. However, stable expression of transgene without disturbing the function of the host genome depends on promoter methylation, transgene copy number and transcriptional activity in integration regions. Recently, new genome-editing tools have made much progress, however little attention has been paid to the identification of genomic safe harbors. The aim of the present study was to evaluate the effect of insertion site, promoter and copy number of transgene on the production of embryos from cattle fibroblast cells following somatic cell nuclear transfer (SCNT). So, three donor vectors were constructed with EGFP gene under control of different promoters. Each vector was integrated into safe and non-safe harbors in the genome using phiC31 integrase. Transgenic clones with a single copy of each vector were isolated. Each clone was analyzed to find site and frequency of integration, expression level and promoter methylation before SCNT, as well as transgene expression level and blastocyst formation rate after SCNT. The data obtained demonstrated that BF5, as a safe harbor, not only showed a stable expression, but also the rate of in vitro-produced embryos from BF5-clones are similar to that of non-transfected cells.
Highlights
Minimum disturbing effect on the function and structure of the target genome[6,7,8]
We attempted to compare the effect of specific integration sites of phiC31as Genomic safe harbors (GSHs) and non-safe harbor sites on EGFP expression from both donor cells and in vitro-derived blastocysts of cattle
According to Yu et al, 17 out of 33 phiC31 pseudo-attP sites identified by this group in the cattle genome were intergenic and only 5 of them were considered as the GSH site (~15% of the total integration sites)[3]
Summary
Minimum disturbing effect on the function and structure of the target genome[6,7,8]. phiC31 integrase is strictly specific to attB sequence (5′-GTGCCAGGGCGTGCCCTTGGGCTCCCGGGCGCG-3′), whereas it does not require conserved attP sequence (5′-CCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGG-3′) for recombination[5]. Much progress has been made in genome editing tools, an important obstacle in the generation of transgenic animals is the gradual decline in transgene expression due to epigenetic modifications[10,15] Based on these complications and the lack of knowledge about GSHs in cattle genome, this study aimed to evaluate the previously described pseudo-attP sites in cattle genome for expression level, promoter resistance against de novo methylation, and the effect of integrated region on the reprogramming following SCNT. By employing such sites as robust GSH, it is possible to utilize specific genome-engineering tools such as Crispr/cas[9] or TALENs for targeting these regions and the efficient generation of transgenic cattle
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