Blastocyst Formation Rate and Transgene Expression are Associated with Gene Insertion into Safe and Non-Safe Harbors in the Cattle Genome

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 nonsafe 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 nontransfected cells.Transgenic farm animals such as goats, sheep, and cows are important biomaterials for biomedical and life science researches, including basic research, protein production, and animal models for human diseases 1,2 . The current approaches to the generation of transgenic animals are often inefficient with a low integration rate and variable expression levels due to random and unstable integration of transgene into chromosomal DNA, position effects and the number of inserted copies 1,3 . In order to overcome these defects, the somatic cell nuclear transfer (SCNT) technology has been developed in combination with the site-specific integration of foreign DNA 4 . SCNT technology can effectively increase the efficiency of the production of transgenic farm animals, but cannot overcome the concern about the integration of foreign DNA, which can be resolved by means of phiC31 integration system 2,3 . PhiC31 is a site-specific integrase derived from actinophage QC31 of Streptomyces and employed as a powerful genetic tool for efficient non-viral delivery of transgene to the host chromosomal DNA 5,6 . The integrase is a member of DNA recombinase family that encoded within the genome of Streptomyces bacteriophage. This enzyme is functional in mammalian cells thus it can mediate recombination reaction between the attP or pseudo-attP site in the host genome and attB site in the plasmid. This serine integrase can specifically integrate the DNA containing attB site into attP and produce DNA element flanked by attL and attR sequences which are no longer identified by phiC31 integrase 2,7 . This system has several useful properties, including unidirectional site-specific integration, stable gene expression, integration into the saf...

Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome

“…Several studies have used strong heterologous promoters to explore whether a predicted locus is GSH [ 3 , 5 , 6 , 10 , 14 , 21 , 24 , 26 , 59 ]. Although the stable expression of the transgene (more than 3 months in the cell lines and at least 1 generation for transgenic animals) has been achieved using strong promoters, it is uneasy to determine whether this durable expression is locus-dependent or promoter-dependent [ 3 , 5 , 6 , 10 , 21 , 24 , 26 ].…”

“…To avoid the effects of negative host-dependent factors on transgene expression, several research projects have tried to find the most appropriate target loci across the genome and to integrate the transgene therein [ 3 – 5 ]. In previous studies, intergenic [ 5 ], intronic [ 3 ], pseudo attP [ 6 ], mCreI [ 7 ], and pMEI [ 8 ] sites have been used as GSH loci to safely host and stably express the transgenes.…”

“…The in vitro evaluation of the long-term expression competency of a transgene integrated into a predicted GSH locus is very important and valuable before generating genetically engineered animals. This is to avoid the possible shutting down of the promoter or silencing of the transgene over time [ 5 , 6 ].…”

“…However, by using weak or tissue-specific promoters, the prediction of the potential GSH regions would be more realistic [ 19 , 29 , 34 , 35 ]. In addition, the investigation of expression profiles of the integrated transgene in a potential GSH locus should be carried out in parallel with the integration of the same transgene in a non-GSH locus [ 6 ]. To our knowledge, there is no report regarding the evaluation of GSH loci using a transgene driven by a weak promoter that is simultaneously integrated both in GSH and non-GSH loci.…”

One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle

“…[11][12][13] When a precomplexed Cas9 protein and sgRNA ribonucleoprotein (RNP) complex is delivered into the target cell or embryo, the Cas9 introduces double-stranded breaks at target DNA sites. 13 At a lower frequency, the double-stranded breaks can undergo homology-directed repair (HDR) in the presence of homologous repair template, or at a higher frequency by error-prone nonhomologous end-joining (NHEJ) pathways 14 Several recent manuscripts highlighted the feasibility of editing bovine genome in somatic cells, [15][16][17][18][19][20] and direct delivery of editors into zygotes 6,[21][22][23] However, no manuscript to date has reported the generation of live calves with novel variants by CRISPR-Cas-mediated HDR editing of bovine genome directly in zygotes. The main goal of this study was to establish a pipeline for (1) rational design and selection of targeting reagents, (2) high throughput screening approaches to evaluate editing outcomes, (3) optimized techniques for embryo injections to maximize HDR outcomes, and (4) ultimately, generation of genome-edited calves with HDR mediated introgression of novel variants at high efficiency.…”