Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 28:44:5C.4.1-5C.4.48.
doi: 10.1002/cpsc.45.

Conditional Manipulation of Gene Function in Human Cells with Optimized Inducible shRNA

Alessandro Bertero  1   2   3 , Loukia Yiangou  1   4 , Stephanie Brown  1   2 , Daniel Ortmann  1   2 , Matthias Pawlowski  1   5 , Ludovic Vallier  1   2   6
Affiliations

Conditional Manipulation of Gene Function in Human Cells with Optimized Inducible shRNA

Alessandro Bertero et al. Curr Protoc Stem Cell Biol. .

Abstract

The difficulties involved in conditionally perturbing complex gene expression networks represent major challenges toward defining the mechanisms controlling human development, physiology, and disease. We developed an OPTimized inducible KnockDown (OPTiKD) platform that addresses the limitations of previous approaches by allowing streamlined, tightly-controlled, and potent loss-of-function experiments for both single and multiple genes. The method relies on single-step genetic engineering of the AAVS1 genomic safe harbor with an optimized tetracycline-responsive cassette driving one or more inducible short hairpin RNAs (shRNAs). OPTiKD provides homogeneous, dose-responsive, and reversible gene knockdown. When implemented in human pluripotent stem cells (hPSCs), the approach can be then applied to a broad range of hPSC-derived mature cell lineages that include neurons, cardiomyocytes, and hepatocytes. Generation of OPTiKD hPSCs in commonly used culture conditions is simple (plasmid based), rapid (two weeks), and highly efficient (>95%). Overall, this method facilitates the functional annotation of the human genome in health and disease. © 2018 by John Wiley & Sons, Inc.

Keywords: gene editing; human pluripotent stem cells; knockdown; shRNA.

PubMed Disclaimer

References

Literature Cited

    1. Avior, Y. , Sagi, I. , & Benvenisty, N. (2016). Pluripotent stem cells in disease modelling and drug discovery. Nature Reviews, Molecular Cell Biology, 17, 170-182. doi: 10.1038/nrm.2015.27
    1. Bertero, A. , Pawlowski, M. , Ortmann, D. , Snijders, K. , Yiangou, L. , Cardoso de Brito, M. , … Vallier, L. (2016). Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development, 143143, 4405-4418. doi: 10.1242/dev.138081.
    1. Boettcher, M. , & McManus, M. T. (2015). Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Molecular Cell, 58, 575-585. doi: 10.1016/j.molcel.2015.04.028.
    1. Boudreau, R. L. , Martins, I. , & Davidson, B. L. (2009). Artificial MicroRNAs as siRNA shuttles: Improved safety as compared to shRNAs in vitro and in vivo. Molecular Therapy, 17, 169-175. doi: 10.1038/mt.2008.231.
    1. Brummelkamp, T. R. , Bernards, R. , & Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550-553. doi: 10.1126/science.1068999.

Publication types