In:
Current Protocols in Stem Cell Biology, Wiley, Vol. 44, No. 1 ( 2018-02)
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.
Type of Medium:
Online Resource
ISSN:
1941-7322
,
1938-8969
DOI:
10.1002/9780470151808.2018.44.issue-1
Language:
English
Publisher:
Wiley
Publication Date:
2018
detail.hit.zdb_id:
2378093-9
Permalink
