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10.1371/journal.pone.0174077 http://scihub22266oqcxt.onion/10.1371/journal.pone.0174077 C5354463!5354463 !28301575     free     free     free Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Warning: imagejpeg(C:\Inetpub\vhosts\kidney.de\httpdocs\phplern\28301575 .jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117       PLoS+One 2017 ; 12 (3 ): e0174077 Nephropedia Template TP {{tp|p=28301575 |t=2017. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system |pdf=|usr=}}{{28301575 }} gab.com Text Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system JO: PLoS One http://www.kidney.de/pget.php?&tw=1&pmid=28301575 #FOAvip In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a ?2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFN? ELISpot assay. The ?2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the ?2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the ?-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor. Twit Text FOAVip Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system ????PLoS One http://www.kidney.de/pget.php?&tw=1&pmid=28301575 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=28301575 #FOAvip English Wikipedia <ref>{{Cite pmid|28301575 }}</ref> Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system #MMPMID28301575 Das K ; Eisel D ; Lenkl C ; Goyal A ; Diederichs S ; Dickes E ; Osen W ; Eichmüller SB PLoS One 2017[]; 12 (3 ): e0174077 PMID28301575 show ga In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a ?2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFN? ELISpot assay. The ?2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the ?2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the ?-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor. |*Clustered Regularly Interspaced Short Palindromic Repeats [MESH] |Animals [MESH] |Cell Line, Tumor [MESH] |Enzyme-Linked Immunosorbent Assay [MESH] |Major Histocompatibility Complex/*immunology [MESH] |Mice [MESH] |Mice, Inbred C57BL [MESH] |Mice, Knockout [MESH]Generation of murine tumor cell lines deficient in MHC molecule surfac(epmc).pdf DeepDyve Pubget Overpricing