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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
Efficient selection of Gal-knockout pig cells for somatic cell nuclear transfer
Luz M Reyes1, Jose L Estrada, Bess Ivary
1Department of Surgery, Transplant Surgery Section, Indiana University School of Medicine, Indianapolis, Indiana.
The Journal of Surgical Research
|July 30, 2013
Summary
This study developed an efficient method for selecting Gal double-knockout (Gal-DKO) cells using fluorescence-activated cell sorting (FACS) and magnetic beads. This improved technique successfully produced healthy Gal-DKO piglets via somatic cell nuclear transfer (SCNT).
Area of Science:
- Animal Biotechnology
- Transgenic Animal Production
- Cell Biology
Background:
- Somatic cell nuclear transfer (SCNT) for generating transgenic animals is hindered by inefficient selection of desired cell genotypes.
- Selecting Gal double-knockout (Gal-DKO) cells for SCNT is particularly time-consuming and inefficient, impacting overall success rates.
Purpose of the Study:
- To develop a highly efficient cell selection system for Gal-DKO cells intended for SCNT.
- To ensure the new selection system does not compromise the efficiency of producing Gal-null pigs.
Main Methods:
- Initial selection of Gal-expression-deficient fetal liver cells using fluorescence-activated cell sorting (FACS) with IB4 lectin.
- Second-round selection employing streptavidin magnetic beads and IB4 lectin biotin for expanded cells.
Main Results:
- Sequential selection achieved a recovery efficiency of 0.04% (FACS) and 0.3% (magnetic beads).
- Selected Gal-DKO cells underwent full reprogramming and were used in SCNT to produce Gal-null piglets.
- Of 48 colonies derived from magnetic bead selection, 44 (91.7%) possessed the Gal-null genotype. Three colonies yielded 17 healthy Gal-DKO piglets from two pregnancies.
Conclusions:
- Sequential FACS and magnetic bead selection provide a highly efficient method for generating Gal-DKO cells.
- The selected Gal-DKO cells are viable and effectively used for SCNT to produce healthy Gal-null piglets.
