Journal: Communications Biology
Article Title: RAISING is a high-performance method for identifying random transgene integration sites
Figure Lengend Snippet: Performance of Rapid Amplification of Integration Site without Interference by Genomic DNA contamination (RAISING). All RAISING products were visualized by electrophoresis on 2% agarose gels. Dotted lines in Sanger sequencing spectra indicate the position of transgene integration sites. a The sensitivity of RAISING was assessed by serially diluting TL-Om1 genomic DNA (an HTLV-1-infected cell line harboring a single copy of HTLV-1) with Jurkat genomic DNA (an HTLV-1 negative cell line). Even extremely diluted samples (resulting in PVL as low as 0.032) could be detected by increasing the cycles in the second PCR of RAISING (left panel). Sanger sequencing analysis of RAISING products confirmed that the same integration site was identified in every dilution (right panel). PVL proviral load. b , c Clonality analyses of infected samples using Sanger sequencing (spectra) vs. high throughput sequencing (HTS, pie charts) showed similar results. b HTLV-1 clonality analysis of an asymptomatic carrier (AC) and an adult T-cell leukemia/lymphoma (ATL) patient. c Bovine leukemia virus (BLV) clonality analysis of an aleukemic (AL) cow and a cow with enzootic bovine leukosis (EBL). d Successful amplification of various transgene-integrated fragments (HTLV-1; HIV-1 human immunodeficiency virus, SIV simian immunodeficiency virus, HBV hepatitis B virus, AdV adenovirus, and low-density lipoprotein receptor knock-in mice Ldlr-mLO-4 and Ldlr-mLO-5) with RAISING. e HBV and AdV integration sites. f Clonality analysis of HIV-1 and SIV-infected cells with HTS analysis. HTS read counts indicate the size of each clone. g RAISING with HTS analysis was used to identify on- and off-target effects in Ldlr-mLO-4 and Ldlr-mLO-5 knock-in mice established by genome editing technology. Chr, chromosome.
Article Snippet: The HTLV-1-infected cell lines TL-Om1, LMY2, ED, KK1, and SLB-1; the HTLV-1-negative acute T-cell leukemia cell line Jurkat (Clone E61: ATCC TIB152); the CD4+ human T-cell line PM1 (3038, NIH AIDS Reagent Program); and the cynomolgus macaque T-cell line HSC-F (JCRB1164) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a 5% CO2 atmosphere , , – .
Techniques: Amplification, Electrophoresis, Sequencing, Infection, Polymerase Chain Reaction, Next-Generation Sequencing, Knock-In, Mouse Assay