SAMN18839303
External Id
Gastroenterology and Hepatology, Erasmus MC
INSDC center name
2021-04-24T00:00:00Z
INSDC first public
2021-04-24T05:02:40.670Z
INSDC last update
SRS8758461
INSDC secondary accession
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
SRS8758461
SRA accession
Primary immortalized human Barrett's cell line
cell line
Functional HOXA13 was removed from BAR-T cells using CRISPR/Cas9-mediated gene editing. A HOXA13 sgRNA targeting exon 1 was cloned into pTLCV2, by ligating two annealed oligonucleotides, i.e. Guide1sgRNA F and R (Extended data table 4). TLCV2 was a gift from Adam Karpf (Addgene #87360)[72]. Following sequence verification, the pTLCV2-HOXA13sgRNA plasmid was packaged into lentiviral particles by cotransfection into HEK293T cells with pSPAX2 and pMD2.G, gifts from Didier Trono (Addgene #12260 and #12259). The supernatant was harvested and ultracentrifuged after which BAR-T cells were transduced. Mixed populations of transduced cells were plated at very low confluence, single cell clones could subsequently be isolated using glass cloning cylinders and low melting point agarose from Sigma-Aldrich, followed by DNA isolation using the Kleargene kit, followed by sequence verification with primers TILHOXA13R3 and Pre-HOXA13-FW2 flanking the sgRNA-site (Extended data table 4). Three cell lines in which both alleles were affected by unique out-of-frame deletions were selected.
genetic modification
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
BAR-T cell line
source_name
BAR-T HOXA13 KO 2
title