Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.
In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc.
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Man
M
Cheng
Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Xubin
X
Jin
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Lili
L
Mu
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Fangyu
F
Wang
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Wei
W
Li
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Xiaoling
X
Zhong
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Xuan
X
Liu
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Wenchen
W
Shen
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Ying
Y
Liu
Department of Cell Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Yan
Y
Zhou
D000818:Animals; D002540:Cerebral Cortex / Q000196:embryology / Q000254:growth & development*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004251:DNA Transposable Elements / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D005260:Female; D047109:Fetal Development; D005333:Fetus; D055786:Gene Knockout Techniques / Q000379:methods*; D051379:Mice; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D058953:Neural Stem Cells; D009462:Neurology / Q000379:methods*; D000071841:PAX6 Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids; D011247:Pregnancy; D015202:Protein Engineering; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
D004251:DNA Transposable Elements; D000071841:PAX6 Transcription Factor; C000605732:Pax6 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse
Sox2; genomic editing; neural progenitor cells