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. The plasmid pJET1.2/blunt was chosen as the main carrier vector by the results of the restriction analysis of the potential components of the genetic engineering structure. The PCR amplification of hLfcDNA was cloned into pJET1.2/blunt to obtain plasmid pJET1.2hLf. Fragments 3'WAP and 5'WAP, cut from the previously obtained intermediate plasmids pTZ5'WAP and pTZ3'WAP, were sequentially cloned intopJET1.2hLf.
Restrictive and PCR assays confirmed the composition of the created plasmid pWAPhLF. Linear gene construction of 4061 bp is cut out by the restriction enzymes EagI (NotI) and ClaI, contains 2136 bp. cDNA hLF,left and right shoulders of homology to the mouse gene for acidic whey protein of the size 945 and 980 bp. respectively. The resulting plasmid (or linearized gene construct) is intended to be used as a template for replacing the coding sequence of the mWAP gene by the cDNA sequence of hLf by the direct homologous recombination mechanism using the CRISPR/Cas9 system.
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