Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor
10.1007/s13770-019-00225-8
- Author:
Yeon Sun KIM
1
;
Gyeong Ryeong KIM
;
Mira PARK
;
Seung Chel YANG
;
So Hee PARK
;
Ji Eun WON
;
Ju Hee LEE
;
Ha Eun SHIN
;
Haengseok SONG
;
Hye-Ryun KIM
Author Information
1. Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea
- Publication Type:Original Article
- From:
Tissue Engineering and Regenerative Medicine
2020;17(1):45-53
- CountryRepublic of Korea
- Language:English
-
Abstract:
METHODS:The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined.
RESULTS:Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting.
CONCLUSION:Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.
