Всероссийский научно-исследовательский институт физиологии, биохимии и питания животных – филиал Федерального государственного бюджетного научного учреждения «Федеральный научный центр животноводства – ВИЖ имени академика Л.К. Эрнста»
ABSTRACT. Based on the genomic sequence of the gene for acidic mouse serum protein (mWAP), primers were chosen for PCR amplification of 5 'and 3'-flanking regions intended for the role of the right and left arms of homology in genetically engineered constructs (GIC) for the purpose of replacing mWAP with coding sequence of a biologically active human protein using the CRISPR / Cas9 system. The 5'WAP PCR amplification was done with the mouse genomic DNA, the promoter region of the mWAP gene, containing the site for the restriction enzyme KpnI immediately before the ATG codon. The reverse primer for PCR amplification contained a site for KpnI, and a site for EagI (NotI) was introduced into the forward primer. 3'WAP PCR amplification was done with the mouse genomic DNA: the region of the 3rd intron of the mWAP gene was chosen to amplify the 3'WAP fragment. In the forward and reverse primers for PCR amplification, sites for the restriction enzymes SalI and ClaI were introduced, respectively. As a result of cloning of the amplifications in pTZ57R / T, the plasmids pTZ5'WAP and pTZ3'WAP were obtained. The reliability of cloning is confirmed by the sequencing of cloned fragments.
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