HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells

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HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells https://t.co/CmQI3nb2a5 @thePeerJ https://t.co/mpwlY4T6vH
Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials and Methods

Generation of LTatCL[M] with target locus homologous arms and Cas9/guide RNA-encoding plasmids

Transfection of Jurkat T-cells and single cell sorting

Activation of silenced HIV-1 promoter in cells with integrated LTatCL[M] in BACH2

Amplifying the junctions of LTatCL[M] integration into BACH2 and AAVS1

Near full-length amplification of integrated vector LTatCL[M]

Quantification of BACH2 mRNA expression

Quantification of BACH2 protein expression

Results

Targeted integration of the 5.3 kb HIV-1-based, dual-fluorophore vector LTatCL[M] into BACH2 and AAVS1 via CRISPR/Cas9-mediated homology directed repair

Silencing of the HIV-1 promoter in Jurkat T-cells with integrated LTatCL[M] in BACH2

Silenced HIV-1 promoters can be reactivated by TNF-α and Romidepsin

Monoclonal single mCherry+ cell lines harbour large internal deletions in LTatCL[M]

Discussion

Conclusion

Supplemental Information

Primers used to amplify the BACH2 and AAVS1 homologous arms, to amplify and sequence the junction between the vector LTatCL[M] and BACH2 or AAVS1, to test for mono- or bi-allelic integration, and to amplify the vector LTatCL[M].

DOI: 10.7717/peerj.10321/supp-1

Additional Information and Declarations

Competing Interests

Karin J. Metzner has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, Tibotec, Bristol-Myers Squibb, and Abbott; the University of Zurich has received research grants from Gilead, Roche and Merck Sharp & Dohme for studies that Karin J. Metzner serves as principal investigator, and advisory board honoraria from Gilead Sciences. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All other authors declare no competing interests relevant to this study.

Author Contributions

Anne Inderbitzin conceived and designed the experiments, performed the experiments, analysed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Yik Lim Kok conceived and designed the experiments, performed the experiments, analysed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Lisa Jörimann performed the experiments, analysed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Audrey Kelley performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Kathrin Neumann performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Daniel Heinzer performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Toni Cathomen conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Karin J. Metzner conceived and designed the experiments, analysed the data, authored or reviewed drafts of the paper, and approved the final draft.

Data Availability

The following information was supplied regarding data availability:

All data is available on Zenodo:

Anne Inderbitzin, Yik Lim Kok, Lisa Meret Joerimann, Audrey Kelley, Kathrin Neumann, Daniel Heinzer, Toni Cathomen, Karin J. Metzner. (5 August 2020). HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells. Zenodo. DOI 10.5281/zenodo.3972946.

Funding

This study was funded by the Swiss National Science Foundation, grant No. 310030_141067/1 to Karin J Metzner and from the Forschungskredit Candoc, grant No. FK-19-032 to Anne Inderbitzin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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