Efficient Gene Knockout via CRISPR

To knockout the gene of interest, a 10–20 pL mixture of 100 ng/µL sgRNA and 200 ng/µL BE3 mRNA were microinjected into putative zygotes 10 h after IVF by using a micromanipulator (TransferMan, Eppendorf, Germany). In the control group, embryos were injected with the same amount of BE3 mRNA without sgRNA. To maximize the editing efficiency of the gene of interest, a cocktail of two or three sgRNAs was microinjected together with BE3 mRNA. Each sgRNA was kept at the same concentration (100 ng/µL).

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Shi Y., Hu B., Wang Z., Wu X., Luo L., Li S., Wang S., Zhang K, & Wang H. (2023). Functional role of GATA3 and CDX2 in lineage specification during bovine early embryonic development. Reproduction (Cambridge, England), 165(3), 325-333.

Publication 2023

TransferMan

Embryos Gene Mrna Pl 100 Zygotes

Corresponding Organization :

Other organizations : Zhejiang University

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Variable analysis

independent variables

Microinjection of 10-20 pL mixture of 100 ng/µL sgRNA and 200 ng/µL BE3 mRNA into putative zygotes 10 h after IVF
Microinjection of a cocktail of two or three sgRNAs (each at 100 ng/µL) together with BE3 mRNA

dependent variables

Editing efficiency of the gene of interest

control variables

Microinjection of the same amount of BE3 mRNA without sgRNA in the control group

controls

Embryos injected with the same amount of BE3 mRNA without sgRNA

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