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10.1038/srep10710 http://scihub22266oqcxt.onion/10.1038/srep10710 C4454075!4454075!26039937     free     free     free Deprecated: Implicit conversion from float 231.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 231.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 231.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 231.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       Sci+Rep 2015 ; 5 (ä): ä Nephropedia Template TP {{tp|p=26039937|t=2015. CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells |pdf=|usr=}}{{26039937}} gab.com Text CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells JO: Sci Rep http://www.kidney.de/pget.php?&tw=1&pmid=26039937 #FOAvip Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways. Twit Text FOAVip CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells ????Sci Rep http://www.kidney.de/pget.php?&tw=1&pmid=26039937 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=26039937 #FOAvip English Wikipedia <ref>{{Cite pmid|26039937}}</ref> CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells #MMPMID26039937 Kimura Y; Oda M; Nakatani T; Sekita Y; Monfort A; Wutz A; Mochizuki H; Nakano T Sci Rep 2015[]; 5 (ä): ä PMID26039937show ga Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways. äCRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem(epmc).pdf DeepDyve Pubget Overpricing