Year: 2020
Type: article
Abstract: The creation of genetically modified animals is becoming an increasingly popular area of scientific and technological progress, primarily for the needs of health and food problems.With the advent of modern genetic engineering methods based on endonucleases, it became possible to obtain animals with high-precision genomic modifications and well-predicted new properties.The creation of self-replicating transgenic animals-bioreactors that secrete biologically active proteins in milk is a promising direction of modern biotechnology.The rabbit is the smallest animal used both in laboratory practice and in animal breeding, from which recombinant pharmaceutical proteins can be obtained on an experimental and industrial scale.Using CRISPR/Cas9 technology, many rabbits have been created in the world -biomodels of human diseases (with gene knockout, transgenic) and producers of human proteins.Rabbits can be effectively used as bioreactors.Whey acidic protein (WAP) is the main protein of whey, its content in rabbit milk is about 15 g / l, which makes the WAP gene promising for replacing the gene of biologically active protein using the CRISPR/Cas9 system.A strategy was developed for site-specific modification of the rabbit WAP gene using CRISPR/Cas9 technology.A plasmid containing the 5' and 3' arms of the WAP rabbit gene homology was created.At the junction of the arms homology, a site for the restriction enzyme EagI was introduced.The reporter protein gene was embedded on the EagI site.A strategy for making double-stranded cuts in the WAP gene was developed and its derivatives encoding Cas9 endonuclease and guiding RNA were obtained.The aim of the study was to evaluate the effectiveness of using the green fluorescent protein (EGFP) gene under the cytomegalovirus promoter in a DNA matrix containing homology arms to the WAP gene as an indicator of homologous directed recombination repair (HDR).
Source: Journal of Livestock Science
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W3107986743