Effects of <italic style="font-style: italic">rpl15</italic> gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development

Tao PENG; Weijun ZHOU; Han LIU; Yanchi ZHAO; Hua LI; Peng ZHANG; Yanhua ZHOU; Liping SHU

doi:10.16016/j.1000-5404.202010180

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Effects of rpl15 gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development

Basic Medicine | 更新时间：2026-01-09

- Effects of rpl15 gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development
- Journal of Third Military Medical University Vol. 43, Issue 8, Pages: 683-691(2021)
- 作者机构：
  
  1.550004 贵阳，贵州医科大学细胞工程生物医药技术国家地方联合工程实验室，基础医学院免疫学教研室，贵州省再生医学重点实验室
  2.550004 贵阳，中国医学科学院成体干细胞转化研究重点实验室
- 作者简介：
- 基金信息：
  
  the Regional Program of National Natural Science Foundation of China(31860325);the Project of Science and Technology Plan of Guizhou Province(黔科合基础【2020】IY092)
- DOI：10.16016/j.1000-5404.202010180
  CLC： R331.2;R394-33;R394.1
- Received：27 October 2020，
  
  Revised：2021-01-04，
  
  Published：30 April 2021
- 稿件说明：
移动端阅览
Tao PENG, Weijun ZHOU, Han LIU, et al. Effects of rpl15 gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development[J]. Journal of Third Military Medical University, 2021, 43(8): 683-691.
DOI：

Tao PENG, Weijun ZHOU, Han LIU, et al. Effects of rpl15 gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development[J]. Journal of Third Military Medical University, 2021, 43(8): 683-691. DOI： 10.16016/j.1000-5404.202010180.

摘要

目的

利用CRISPR/Cas9技术敲除斑马鱼

rpl15

基因，探讨

rpl15

基因对斑马鱼红系造血发育的影响。

方法

针对斑马鱼

rpl15

基因设计并制备gRNA(guide RNA)，将gRNA与Cas9蛋白的混合物显微注射到单细胞期斑马鱼受精卵中，提取72 h胚胎基因组DNA，PCR扩增出目的片段，T7E1限制性酶切法检测基因打靶情况，随后进行测序分析确定编辑效果。通过O-dianisidine染色分析血红蛋白合成情况，中性粒细胞染色分析中性粒细胞生成情况，利用Real-time PCR检测

p53

、

mdm2

、

hsp70

和造血相关转录因子基因的表达变化。

结果

成功制备了

rpl15

gRNA

并筛选得到

rpl15

突变体。

rpl15

基因敲除的幼鱼出现体长缩短、尾部弯曲、头部和眼睛较小及心包水肿的表型；造血相关谱系染色结果显示

rpl15

突变体血红蛋白合成明显减少，但中性粒细胞生成未受影响；

rpl15

突变体

p 53

和

mdm2

的mRNA表达均显著升高(

0.01，

0.05

)，

α-globin

、

gata1

和

hsp70

的mRNA表达均显著下调(

0.01，

0.05，

0.05)，除红系外的造血相关转录因子的mRNA表达与对照组相比均无明显差异。

结论

利用CRISPR/Cas9技术成功编辑了斑马鱼

rpl15

基因，突变体产生了类似先天性纯红细胞再生障碍性贫血疾病的表型。同时表明

p53

与

hsp70

可能参与调控突变体表型的形成。

Abstract

Objective

To determine the effect of knocking out zebrafish

rpl15

gene by CRISPR/Cas9 editing technique on zebrafish erythroid hematopoietic d

evelopment.

Methods

Guide RNA was designed and prepared for

rpl15

gene of zebrafish and the mixture of gRNA and Cas9 protein was microinjected into zebrafish zygote at single cell stage. Genomic DNA was extracted after 72 h and the target fragment was amplified by PCR. Editing effect was evaluated by T7E1 restriction enzyme digestion of the PCR products followed by DNA sequencing. Hemoglobin synthesis was analyzed by O-dianisidine staining

neutrophil production was analyzed by neutrophil staining

and the expression of

p53

mdm2

hsp70

and hematopoiesis related transcription factor genes was detected by real-time PCR.

Results

The

rpl15

gRNA was successfully prepared and the

rpl15

mutants were selected. The results showed that zebrafish

rpl15

gene knockout juveniles showed a phenotype of shortened body length

curved tail

smaller head and eyes

and pericardial edema. Hematopoietic lineage staining showed that hemoglobin synthesis in

rpl15

mutant was significantly decreased

but neutrophil production was not affected. The mRNA expression levels of

rpl15

mutant

p53

and

mdm2

were significantly increased (

0.01

0.05)

while those of

α-globin

gata 1

and

hsp70

were obviously down-regulated (

0.01

0.05

0.05). There were no significant differences between the

rpl15

mutant and the control group in the mRNA expression levels of hematopoiesis-related transcription factors except erythroid-related ones.

Conclusion

The

rpl15

gene of zebrafish is successfully edited by CRISPR/Cas9 technique

and the mutant produces a phenotype similar to Di

amoncl-Blackfan anemia disease. At the same time

it is suggested that

p53

and

hsp70

may be involved in regulating the formation of mutant phenotype.

关键词

Keywords

references

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⁰

Effects of rpl15 gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development

DOI：10.16016/j.1000-5404.202010180

摘要

Abstract

关键词

Keywords

references