Chinese scientists realize targeted genetic screening at the individual level of mice

recent years, CRISPR-Cas9 technology-mediated mutation screening has been widely used in mice and human cells, and the realization of efficient individual-level genetic screening in mice is of great significance to the study of mammalian functional genomes.
Li Jinsong of the Center for Excellence and Innovation in Molecular Cell Science of the Chinese Academy of Sciences (Institute of Biochemistry and Cell Biology) and Yan Weiguo's research team worked together to apply semiclonal technology mediated by lone male monolithic embryonic stem cells ("artificial sperm cells"), in combination with CRISPR-Cas9-mediated genes Editing technology successfully realized the individual level genetic screening of bone development-related genes in mice, and revealed that the Irx5 gene as an important regulatory factor in bone development, by inhibiting PPAR gamma molecules to promote bone differentiation and inhibit lipogen differentiation, opened up a new way for the study of key gene function in mouse development. The study was published online July 2 in Plos Biology, the American Public Library of Science.
Li Jinsong, a researcher, told China Science Daily that researchers screened 72 potential bone development-related genes based on high-flux sequencing data from the in-body bone differentiation process, created a 216-guide RNA (sg) RNA)'s throosome stem cell bank, or "artificial sperm cell bank", each of which typically carries a "sgRNA navigator" for different genes, precisely locating and editing the target gene. "We injected these 'artificial sperm' carrying modified genes one by one into mice's MII. phase ovary cells, and in less than a month we obtained more than 400 semi-cloned mice carrying different mutation genes, " Li explained. "
After the mice were born, we began to analyze the overall staining of their bones, scoring each mouse according to indicators such as bone size, calcification, skull, long bone, vertebrae morphology, and so on. At the same time, we used the 'sgRNA navigator' carried by each mouse to sequence the 'target gene' carried by mice with abnormal bone development to determine whether it had a genetic mutation. "
" we selected four key genes involved in bone development regulation from 72 candidate genes. "Of these four key genes, genes Zic1 and Clep11a have been confirmed by other studies to be involved in bone development regulation, while the function of genes Rln1 and Irx5 in bone development in mice is not yet clear. To test their regulatory mechanisms in bone development in mice, we constructed mouse models of the genes Rln1 and Irx5 to verify the role of these two genes in bone development, and found that mice that knocked out the Rln1 gene had smaller bones at birth and no significant differences in adult life; Further molecular mechanism studies have shown that IRX5 promotes bone differentiation while inhibiting fat production by inhibiting the activation of the PPAR gamma path. The theoretical
the study is that for the first time, targeted genetic screening for individual bone development in mice was achieved. Referring to the value of future clinical applications, He said, "We hope that our basic research will provide theoretical support for clinical services, drug development and clinical applications for osteoporosis," he said. For
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