Preparation of Nucleic Acid Solutions for Endonuclease Induced Modification of the Mouse or Other Mammalian Genomes V1

The Transgenic Core has used Cas9, ZFNs, and TALENs, to target more 500 genes in mice and rats.In the current era, all projects involve gene knockins, such as floxed alleles, recombinase knockins, reporter knockins, cDNA knockins to the ROSA26 locus. Simple gene knockouts are not in demand as they were when CRISPR/Cas9 technology was reported n 2013. It is essential to protect sensitive RNA components from degradation during zygote microinjection and/or electroporation. All materials mixed with gRNA or mRNA components, such as Cas9 protein (used to prepare ribonucleotide protein complexes), DNA donors (such as oligonucleotides, long single stranded DNA donors, and double stranded circular plasmids), and AAV donors must be resuspended in a buffer that is free from endonucleolytic activity.Fine particulate contaminants can be reduced in microinjection solutions by judicious use of 0.02 µm filters. The suggestions below are based our successful experience in the production of genetically engineered mice, rats, and cultured cells.