Goat CDK2 (Cyclin-dependent kinases 2) gene knockout vector and construction method thereof

Abstract

The invention discloses a goat CDK2 (Cyclin-dependent kinases 2) gene knockout vector and a construction method thereof. A CRISPR / Cas9 system is adopted; an SgRNA segment of a CDK2 gene is designed at first and an SgRNA nucleotide sequence is synthesized; a plasmid PYSY-sgRNA for expressing SgRNA and Cas9D10A at the same time is constructed; the plasmid PYSY-sgRNA is connected and transformed to an escherichia coli DH5alpha competent cell; finally, a transformant is verified; restriction enzyme digestion and sequencing identification prove that the construction of the CDK2 gene knockout vector is accurate. The vector constructed by CRISPR / Cas9 is adopted, and a theoretical basis is provided for subsequently obtaining a goat CDK2 gene deletion type cell line and researching a molecular mechanism of cell apoptosis molecules triggered by mycoplasma pneumonia infection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a goat CDK2 gene knockout vector and a construction method thereof. Background technique [0002] The drive of CDKs can complete cell cyclin-dependent protein kinase-2 (Cyclin-dependentkinases 2, CDK 2) is one of the family members of CDK. The biological function of CDK2 is mainly involved in cell cycle regulation, which can be activated by cyclin E (cyclin E) and cyclin A (cyclin A) at different stages of the cell cycle, thereby promoting the transcription of a series of cell cycle-related genes , the dual role of initiating DNA replication and inducing mitosis. [0003] The CRISPR / Cas9 system is one of the immune mechanisms that bacteria have evolved under the long-term selection pressure of phages to effectively resist the invasion of foreign DNA. In bacteria, the CRISPR cluster is transcribed into precrRNA under the control of its leader region, and ...

AI Technical Summary

Problems solved by technology

[0004] At present, ZFNs or TALENs are used for gene knockout. To edit each gene site, two nucleases need to be designed and assembled. The construction technology is difficult and the construction and assembly time is relatively long.

In addition, traditional gene knockout mainly uses the principle of gene recombination to lose the function of the target gene through insertion mutation and targeting technology.

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