CRISPR/Cas9技术敲除斑马鱼lkb1基因及表型分析

梁森; 李帅庭; 袁志; 黄慧哲

doi:10.16016/j.1000-5404.201902114

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CRISPR/Cas9技术敲除斑马鱼lkb1基因及表型分析

基础医学 | 更新时间：2026-01-09

- CRISPR/Cas9技术敲除斑马鱼lkb1基因及表型分析
- Knockout and phenotype analysis of lkb1 gene in zebrafish by CRISPR/Cas9 technology
- 第三军医大学学报 2019年41卷第15期页码：1445-1452
- 作者机构：
- 作者简介：
- 基金信息：
- DOI：10.16016/j.1000-5404.201902114
  中图分类号：
- 收稿：2019-02-24，
  
  纸质出版：2019-08-15
- 稿件说明：
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梁森, 李帅庭, 袁志, 等. CRISPR/Cas9技术敲除斑马鱼lkb1基因及表型分析[J]. 第三军医大学学报, 2019,41(15):1445-1452.

Sen LIANG, Shuaiting LI, Zhi YUAN, et al. Knockout and phenotype analysis of lkb1 gene in zebrafish by CRISPR/Cas9 technology[J]. Journal of Third Military Medical University, 2019, 41(15): 1445-1452.
梁森, 李帅庭, 袁志, 等. CRISPR/Cas9技术敲除斑马鱼lkb1基因及表型分析[J]. 第三军医大学学报, 2019,41(15):1445-1452. DOI： 10.16016/j.1000-5404.201902114.

Sen LIANG, Shuaiting LI, Zhi YUAN, et al. Knockout and phenotype analysis of lkb1 gene in zebrafish by CRISPR/Cas9 technology[J]. Journal of Third Military Medical University, 2019, 41(15): 1445-1452. DOI： 10.16016/j.1000-5404.201902114.

摘要

Abstract

Objective

To knock out lkb1 gene in zebrafish by CRISPR/Cas9 technique

observe and verify homozygote phenotype

and preliminarily explore the effect of lkb1 on the development of renal tubule.

Methods

The gRNA targeting lkb1gene was designed and synthesized. The gRNA and Cas9 mRNA were injected into the wild-type cell-phase embryo yolk by microinjection technique. The high-efficiency gRNA was screened by T7E1 digestion and sequencing. The F0 mutants and heritable mutants were screened by sequencing. Wild-type fish and heritable mutant fish were used to hybridize to produce F1 hybrid heterozygous mutants

and F1 hybrid heterozygous mutants were identified by sequencing. Then F2 generation homozygous juveniles were produced by self-crossing of F1 heterozygotes. Homozygous phenotypes were identified

and homozygous phenotypes were verified by qPCR and Western blot analysis.

Results

The zebrafish lkb1 gRNA and Cas9 mRNA were prepared

and 3 heterozygotes with frameshift mutations were obtained. It was found that the zebrafish Lkb1 knockout homozygous juveniles violently swayed at the head and tail on the 5th day after fertilization

and with missed swim bladder

bended body axis

fast yolk consumption

dim liver

and edematous intestinal tract

and most of them died on the 9th day. The mRNA levels of homozygous g6pase

pdk2b and pcpck1 were significantly increased

while lkb1 was not expressed at all. The mRNA levels of homozygous renal markers

trmp7

slc12a1 and slc12a3 were also obviously elevated.

Conclusion

Knockout of zebrafish lkb1 gene by CRISPR/Cas9 technology results in liver failure

edematous intestinal tract

bended body axis

missed swim bladder

and early lethal phenotype. The knockout also shows effect on the development of renal tubule.

关键词

Keywords

references

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