Designing of a DNA matrix for transgene integration into the bovine beta-lactoglobulin gene locus using CRISPR/Cas9 technology
Beta-lactoglobulin (BLG) is the main protein in milk serum in almost all mammals, with the exception of rodents and primates. Regulatory regions of the beta-lactoglobulin gene in ruminants (sheep, goats, and cattle) as part of genetic constructs provide tissue - specific expression of recombinant protein in the mammary gland and have been actively used in genetic engineering since the beginning of the era of creating transgenic animals. To work effectively with the CRISPR/Cas9 genomic editing method, it is necessary to know the exact DNA sequence of the target gene: this is necessary both for creating a DNA matrix for homologous recombination and for the targeted accuracy of guide RNAs. A polymorphic variant of the bovine BLG gene was identified, whose sperm was used to fertilize cow oocytes in vitro. The aim of this work was to create a plasmid containing 5’ - and 3’ - arms of homology (ha) to the bovine BLG gene. Based on ??TZ57R/T, the pTZhaBLG plasmid was obtained, which has a unique site for EagI restriction at the junction of the homology arms. A fragment containing a biologically active protein gene can be embedded in the resulting plasmid at this restriction site. We created the pBLGcmvEGFP plasmid containing the green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter: protein expression can serve as a reliable indicator of successful integration of the transgene into the genome. The resulting plasmids in circular or linearized form are intended for site-specific integration by homologous recombination repair into the BLG gene using CRISPR/Cas9 components.