The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked-out mice...

Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination-activating gene 1) knocked-out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real-time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked-out blastocyst. Our results showed that the expression of trophectoderm genes (-TEAD-4 and Cdx2), pluripotency genes (Nanog and Oct-4) and imprinting gene (H19) in the Rag1 knocked-out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked-out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses.