Establishment of paired box gene 2 knockout mice with CRISPR/Cas9 gene targeting technology

WANG Min; ZHAO Min; SHI Xiang-Cheng; LI Rong-Shan

doi:10.16098/j.issn.0529-1356.2020.04.023

PDF(7593 KB)

Acta Anatomica Sinica ›› 2020, Vol. 51 ›› Issue (4) : 613-617. DOI: 10.16098/j.issn.0529-1356.2020.04.023

Technology and Methodology

Establishment of paired box gene 2 knockout mice with CRISPR/Cas9 gene targeting technology

WEI Hong-en¹ WANG Min¹ ZHAO Min¹SHI Xiang-cheng² LI Rong-shan^3*

Author information +

History +

Abstract

Objective To establish paired box gene 2(Pax2) gene knockout mice by clusterd regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 (Cas9) technology with double nicking method so as to provide an animal model for studying the role of Pax2 gene in multiple development. Methods The sgRNA was designed according to the Pax2 gene sequence, and the designed sgRNA and Cas9 were microinjected into the fertilized eggs of C57BL/6J mice in vitro. F0 generation mice were genetically sequenced to identify genotypes after birth, and a total of 8 F0 generation mice were obtained. The F0 mice were mated with wild C57BL/6J mice to obtain F1 mice, and the mice successfully knocked out were successfully mated with C57BL/6J mice to obtain stable Pax2 knockout mice. Results The stably propagated Pax2 heterozygous knockout mice were successfully obtained, and the Pax2 gene was deleted by 1628 bp. HE staining showed that the number of glomeruli in knockout mice was significantly reduced. Western blotting result showed that the expression of Pax2 protein in renal cortex of knockout mice was reduced compared with that in wild type mice. Conclusion The Pax2 heterozygous knockout mice can be successfully constructed by using CRISPR/Cas9 technology, which lays a foundation for further study of the role of Pax2 gene.

Key words

Paired box gen 2 / CRISPR/Cas9 techonology / Gene knockout / Renal developmental disorder / Western blotting / Mouse

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

WEI Hong-en WANG Min ZHAO Min SHI Xiang-cheng LI Rong-shan. Establishment of paired box gene 2 knockout mice with CRISPR/Cas9 gene targeting technology[J]. Acta Anatomica Sinica. 2020, 51(4): 613-617 https://doi.org/10.16098/j.issn.0529-1356.2020.04.023

References

［1］ Hou XM, Chen X, Wang YL. The role of in regulation of kidney development and kidney disease［J］. Hereditas, 2011, 33(9):931-938. (in Chinese)

侯晓明，陈星，王玉林. Pax2在肾脏发育和肾疾病中的调控作用［J］. 遗传, 2011, 33(9): 931-938.

［2］ Fotaki V, Price DJ, Mason JO. Newly identified patterns of Pax2 expression in the developing mouse forebrain［J］. BMC Dev Biol,2008, 8: 79.

［3］ Torres M, Gomez-Pardo E, Dressler GR, et al. Pax-2 controls multiple steps of urogenital development［J］. Development,1995, 121 (12): 4057-4065.

［4］ Namm A, Arend A, Aunapuu M. Expression of Pax2 protein during the formation of the central nervous system in human embryos［J］. Folia Morphol (Warsz),2014, 73 (3): 272-278.

［5］ Miyazawa T, Nakano M, Takemura Y, et al. A case of renal-coloboma syndrome associated with mental developmental delay exhibiting a novel PAX2 gene mutation［J］. Clin Nephrol,2009, 72 (6): 497-500.

［6］ Wefers AK, Haberlandt C, Surchev L, et al. Migration of interneuron precursors in the nascent cerebellar cortex［J］. Cerebellum,2018, 17 (1): 62-71.

［7］ Fauquier T, Romero E, Picou F, et al. Severe impairment of cerebellum development in mice expressing a dominant-negative mutation inactivating thyroid hormone receptor alpha1 isoform［J］. Dev Biol,2011, 356 (2): 350-358.

［8］ Fallahi-Sichani M, Soleimani M, Najafi SM, et al. In vitro differentiation of cord blood unrestricted somatic stem cells expressing dopamine-associated genes into neuronlike cells［J］. Cell Biol Int,2007, 31 (3): 299-303.

［9］ Nornes HO, Dressler GR, Knapik EW, et al. Spatially and temporally restricted expression of Pax2 during marine neurogenesis［J］. Development,1990, 109 (4): 797-809.

［10］ Chandrasegaran S, Carroll D. Origins of programmable nucleases for genome engineering［J］. J Mol Biol,2016, 428 (5 Pt B): 963-989.

［11］ Pattanayak Ⅴ, Ramirez CL, Joung JK, et al. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection［J］. Nat Methods,2011, 8 (9): 765-770.

［12］ Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems［J］. Science,2013, 339 (6121): 819-823.

［13］ Hussain W, Mahmood T, Hussain J, et al. CRISPR/Cas system: a game changing genome editing technology, to treat human genetic diseases［J］. Gene,2019, 685：70-75.

［14］ Jinek M, Chylinski K, Fonfara Ⅰ, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity［J］. Science,2012, 337 (6096): 816-821.

［15］ Zheng W, Gu F. Progress of application and off-target effects of CRISPR/Cas9［J］. Hereditas, 2015, 37(10): 1003-1010. (in Chinese)

郑武，谷峰.CRISPR/Cas9的应用及脱靶效应研究进展［J］.遗传, 2015, (10): 1003-1010.

［16］ Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases［J］. Nat Biotechnol,2013, 31 (9): 827-832.

［17］ Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification［J］. Nat Biotechnol,2014, 32 (6): 577-582.

［18］ Shen B, Zhang W, Zhang J, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects［J］. Nat Methods,2014, 11 (4): 399-402.

［19］ Li ZhY, Huang ChC, Sun XH, et al. Rat models of AR and IRS2 knockout genes were prepared using CRISPR/Cas9 technology［J］. Journal of Tropical Medicine, 2018, 18(9): 1143-1146. (in Chinese)

李忠义，黄垂灿，孙秀红，等. 利用CRISPR/Cas9技术制备敲除AR和IRS2基因大鼠［J］. 热带医学杂志, 2018, 18(9): 1143-1146.