SAMN18839297
External Id
Gastroenterology and Hepatology, Erasmus MC
INSDC center name
2021-04-24T00:00:00Z
INSDC first public
2021-04-24T05:02:49.670Z
INSDC last update
SRS8758490
INSDC secondary accession
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
SRS8758490
SRA accession
Primary immortalized human squamous esophageal cell line
cell line
The human HOXA13 gene including its single intron was amplified using Q5 polymerase from gDNA using primers (AgeI HoxA13 F; GGTGGTACCGGTGCCACCATGACAGCCTCCGTGCTCCT, and XbaI HoxA13 R; ACCACCTCTAGATTAACTAGTGGTTTTCAGTT) and cloned into pEN_TmiRc3 using AgeI and XbaI restriction sites, a gift from Iain Fraser (Addgene (Cambridge, USA) #25748). Subsequently, the HOXA13 insert was transferred into pSLIK-Venus, using a Gateway reaction. pSLIK-Venus was a gift from Iain Fraser (Addgene #25734). LGC Genomics (Teddington, UK) sequenced both plasmids. Next, plasmids were packaged into lentiviral particles following transfection in HEK293T cells with third generation packaging plasmids. The supernatant was collected and ultracentrifuged. EPC2-hTERT cells were transduced with the virus and Fluorescence-Activated Cell Sorted (FACS) for YFP (pSLIK-Venus) positive cells on the BD FACSCantoTM II that was bought from BD Biosciences (San Jose, USA). These cells were grown and analyzed as a cell pool. HOXA13 was induced by the addition of 1,25 µg/ml doxycycline to the culture medium. Overexpression was determined by qPCR according to scientific standards.
genetic modification
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
EPC2-hTERT cell line
source_name
EPC2-hTERT HOXA13 1
title