A pair of Talen recognition sequences targeting zebrafish forkhead box n1 gene and its mRNA preparation method

Abstract

The invention discloses a pair of Talen recognition sequences targeting the zebrafish Forkhead box n1 gene and a method for preparing mRNA for knocking out the zebrafish Forkhead box n1 gene. The gene sequences of the Talen recognition sequence pair are respectively shown in the sequence table SEQ ID NO.1 and SEQ ID NO.2, and the preparation method of the mRNA comprises the following steps a) assembling and synthesizing the Talen recognition sequence described in claim 1 Right; b) according to the Talen recognition sequence pair described in a), construct an expression vector targeting the gene knockout effector protein Talen of the Forkhead box n1 gene; c) transfect the vector obtained in step b) by liposome, Detecting protein expression in 293T cells; d) transcribing the vector obtained in step b) in vitro to obtain the mRNA for targeted knockout of the zebrafish Forkhead box n1 gene. The mRNA obtained by the method of the present invention can be used to knock out the zebrafish Forkhead box n1 gene to prepare a research model of zebrafish thymus development. Compared with the prior art, the method has the advantages of low cost, simple design, high success rate and obvious effect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for knocking out the zebrafish Forkheadbox n1 gene by using gene targeting technology. Background technique [0002] The thymus is a key central organ for the maturation of T cells, which can secrete a variety of thymus hormones and hormone substances. The Forkhead Box (Fox) transcription factor family is a group of winged helix / forkhead transcription factor families. More than 20 Forkhead genes have been found in humans. This transcription factor plays a key role in embryonic development, especially in endoderm developmental organs Regulatory role Forkhead box n1 is involved in the formation, development, maturation and degeneration of the thymus in mammals. Mutations or deletions of the Foxn1 gene in humans and mice can cause severe developmental disorders or deletions of the thymus (Li Hongran and Zhang Lianjun et al., 2010). In the study of zebrafish thy...

AI Technical Summary

Problems solved by technology

[0004] Traditional gene knockout technology relies on the random exchange of homologous chromosomes that occur naturally in cells, but in somatic cells, the efficiency of gene homologous recombination is particularly low (less than 10 -6 ), which increases the workload of actual operation and limits the application of this technology

[0005] Gene knockout caused by RNA interference: RNA interference (RNA interference, RNAi) is an RNA-dependent gene silencing phenomenon, which is a sequence-specific post-transcriptional gene expression silencing induced by double-stranded RNA molecules at the mRNA level. Under the action, a series of siRNA (small interference RNA) with a length of 21-22 nt can be produced. The siRNA molecular nuclease and helicase combine to form the RNA-induced silencing complex (RISC). Strand siRNA unwinds, using the single-stranded siRNA inside the RISC, recognizes the complementary target RNA through base pairing, cleaves the target RNA, and degrades it by RNase, resulting in the silencing of the target gene. Therefore, by introducing dsRNA molecules into cells Within, specifically degrade the homologous mRNA in the cell, and block the expression of the endogenous gene to inactivate the gene can also achieve gene knockout, but RNAi cannot act on all genes and certain cell types, and there are positions Effects, Temporary and Drawbacks of Incomplete Knockouts

Therefore, RNAi cannot completely replace traditional gene knockout techniques

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