Goat TLR4 gene knock-out vector and construction method thereof

Abstract

The invention discloses a goat TLR4 gene knock-out vector and a construction method thereof. The construction method comprises the following steps: firstly, designing an sgRNA fragment of the TLR4 gene by adopting a CRISPR/cas9 system, synthesizing an sgRNA nucleotide sequence, constructing and simultaneously expressing the sgRNA and plasmid PYSY-sgRNA of Cas9 D10A, connecting and transforming to an Escherichia coli DH5 alpha competent cell, and verifying the transformant; and judging and proving by enzyme digestion and sequencing that the TLR4 gene knock-out vector is constructed correctly. The invention adopts the CRISPR/cas9 for constructing the vector, and provides a theoretical basis for subsequently acquiring a goat TLR4 gene deletion type alveolar epithelial cell system, and studying the immune response molecular mechanism of mycoplasma pneumonia infection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a goat TLR4 gene knockout vector and a construction method thereof. Background technique [0002] Toll-like receptors (TLRs) are an important pattern recognition receptor in the innate immune system, which can selectively recognize pathogenic microorganisms and initiate innate immunity, and play an important role in host innate and acquired immunity. TLR4, one of the important members of the TLRs family, is the most important pattern recognition receptor of endotoxin lipopolysaccharide (LPS), and plays a key role in mediating the host response of negative bacteria and their LPS. [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 lead...

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.

[0005] However, there is no report on the knockout of TLR4 gene and its related functions in goats

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