I am new to CRISPR Knock In. I want to clone a gene into ROSA 26 donor plasmid to Knock In a gene into the mouse genome. I have selected the addgene plasmid # 74285. But I cannot figure out where to clone in b/w left and right arms of ROSA 26.
Hi, hello, I am Chne Jing. You need to BLAST your ROSA26 plasmid in the mouse ROSA26 gene sequence and look for the restriction sites and open reading frames in the sequence. You will find some different places on the plasmid, including the clone location. CRISPR KI mice not only need to construct efficient plasmid vectors but also microinject the vectors into mouse single-cell embryos and transplant these cells into pseudo-pregnant mice. Finally, the offspring mice are genotyped to screen out the target mice. Our laboratory is also using CRISPR-edited cell lines and mice, but we are only editing the cell genes ourselves, and the CRISPR mice are delivered to the CRISPR/Cas9 PlatformCB biotech company. Hope it helps you
https://www.creative-biogene.com/crispr-cas9/service/rosa26-knockin-mouse.html
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