scholarly journals Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

10.3791/58467 ◽  
2018 ◽  
Author(s):  
Dan Zhou ◽  
Lei Tan ◽  
Jian Li ◽  
Tanbin Liu ◽  
Yi Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Polymerase Chain Reaction ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Southern Blotting ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Use of two gRNAs for CRISPR/Cas9 improves bi-allelic homologous recombination efficiency in mouse embryonic stem cells

genesis ◽  
2018 ◽  
Vol 56 (5) ◽  
pp. e23212 ◽  
Author(s):  
Sandra Acosta ◽  
Luciano Fiore ◽  
Isabel Anna Carota ◽  
Guillermo Oliver
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Recombination Efficiency

scholarly journals Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

1992 ◽  
Vol 12 (6) ◽  
pp. 2769-2776 ◽  
Author(s):  
A C Davis ◽  
M Wims ◽  
A Bradley
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Selectable Marker ◽  
Embryonic Stem ◽  
Chain Reaction ◽  
Genetic Changes ◽  
Replacement Vector ◽  
Hprt Locus ◽  
Polymerase Chain ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.

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