A simple polymerase chain reaction screen for homologous targeting in embryonic stem cells

2004 ◽  
Vol 332 (2) ◽  
pp. 401-403 ◽  
Author(s):  
Jeffrey McDermott ◽  
Ying Zhao ◽  
Brian Sauer
Keyword(s):  
Stem Cells ◽  
Polymerase Chain Reaction ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Chain Reaction ◽  
Polymerase Chain

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.

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.

Differentiation of mesenchymal stem cells into neuron-like cells using composite 3D scaffold combined with valproic acid induction

2017 ◽  
Vol 32 (6) ◽  
pp. 702-715 ◽  
Author(s):  
Sadegh Ghorbani ◽  
Taki Tiraihi ◽  
Masoud Soleimani
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Polymerase Chain Reaction ◽  
Mesenchymal Stem Cells ◽  
Lactic Acid ◽  
Reverse Transcription ◽  
Neural Regeneration ◽  
Poly Lactic Acid ◽  
Chain Reaction ◽  
Polymerase Chain

The nervous system has little capacity for self-repair after injury because neurons cannot proliferate owing to lack of suitable microenvironment. Therefore, neural tissue engineering that combines neural stem, scaffolds, and growth factors may improve the chance of restoration of damaged neural tissues. A favorable niche for neural regeneration would be both fibrous and electrically conductive scaffolds. Human Wharton jelly-derived mesenchymal stem cells were seeded on wet-electrospun 3D scaffolds composed of poly lactic acid coated with natural polymers including alginate and gelatin, followed by a multi-wall carbon nanotube coating. The results show that a wet-electrospun poly lactic acid scaffold at a concentration of 15% w/v had higher porosity (above 80%) than other concentrations. Moreover, the coated scaffold supported the growth of human Wharton jelly-derived mesenchymal stem cells in 3D culture, and were incubated for 21 days with 1 mM valproic acid as the inducer resulted in improvement in human Wharton jelly-derived mesenchymal stem cells differentiation into neuron-like cells immunoreactivity to nestin, Map2, and neuron specific enolase (NSE), which were also consistent with reverse transcription polymerase chain reaction (RT-PCR) and quantitive Reverse transcription polymerase chain reaction (qRT-PCR) results. The conclusion is that the 3D composite nanofiber poly lactic acid scaffold improved the transdifferentiation of human Wharton jelly-derived mesenchymal stem cells into neuron-like cells.

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