Rapid screening method applied to model animal zebrafish transgenosis

Abstract

The invention provides a rapid screening method applied to model animal zebrafish transgenosis. The method includes the following steps of 1, building a gene expression vector containing a strong promoter, an objective gene and a label gene; 2, designing a specific target spot aiming at a target gene, and obtaining a targeted SgRNA sequence; 3, based on the specific target spot, building a homologous recombinant vector; 4, injecting a homologous recombinant vector section, mRNA obtained through in-vitro transcription synthesis of the SgRNA sequence and nCas9-mRNA obtained through in-vitro transcription synthesis together into a zebrafish zygote; 5, firstly, screening out zebrafishes lack of a target gene function, then conducting second screening through the label gene, obtaining a label gene expression individual, and therefore obtaining the expression individual which expresses the objective gene. Through the method, early juvenile zebrafishes can be screened, and the purpose of quickly establishing a transgenic model is achieved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a rapid screening method applied to zebrafish transgenes. Background technique [0002] In the study of gene function, the deletion and overexpression study of the target gene is the main research approach. At present, there are three main methods for gene expression in zebrafish: one is to synthesize the mRNA of the target gene in vitro, and introduce it into the zebrafish embryo to achieve the purpose of expression through microinjection. The uneven distribution in cells leads to inaccurate expression patterns. At the same time, mRNA is easily degraded in zebrafish, so it can only achieve transient overexpression, and cannot obtain the spatiotemporal expression pattern of overexpressed genes. The second is to construct a gene expression vector and use non-homologous end joining to integrate the vector into the genome. This method is inefficient, time-consuming, and requires ...

AI Technical Summary

Problems solved by technology

At present, there are three main methods for gene expression in zebrafish: one is to synthesize the mRNA of the target gene in vitro, and introduce it into the zebrafish embryo to achieve the purpose of expression through microinjection. Uneven distribution in cells leads to inaccurate expression patterns. At the same time, mRNA is easily degraded in zebrafish, so it can only achieve transient overexpression, and cannot obtain the spatiotemporal expression pattern of overexpressed genes.

The second is to construct a gene expression vector and use non-homologous end joining to integrate the vector into the genome. This method is inefficient, time-consuming, and requires a large number of zebrafish screening. This method is a common strategy for cells. Zebrafish don't live up to expectations

The third is the transgene mediated by transposon, that is, by constructing a transposon vector to transfer the target gene into the host to achieve the purpose of transgenesis. This method has high efficiency, but due to the transposon Random insertions, often causing unintended mutations, and requiring extensive screening efforts

Although homologous recombination-mediated gene knock-in has been reported in several cells and organisms, such as human pluripotent stem cells and mice, there is still room for progress in gene knock-in, especially in improving gene recombination efficiency and applicability in various animal models and cells

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