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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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Array-based high-throughput screening in mouse embryonic stem cells with shRNAs.

Chia-Hui Wang1, Nianhan Ma2, Yu-Tsen Lin1,3

  • 1Genomics Research Center, Academia Sinica, Taipei, Taiwan.

Current Protocols in Stem Cell Biology
|February 11, 2014
PubMed
Summary

This study presents a rapid method using short-hairpin RNA (shRNA) lentivirus screening to discover regulators of embryonic stem cell (ESC) renewal. The technique efficiently identifies factors influencing ESC pluripotency and expansion.

Keywords:
RNA interferenceembryonic stem cellshigh-throughput screeningshort-hairpin RNA

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Area of Science:

  • Stem Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Embryonic stem cells (ESCs) possess self-renewal and pluripotency, making them vital for developmental biology and regenerative medicine.
  • Identifying regulators of ESC renewal is crucial for understanding pluripotency and controlling cell expansion.
  • High-throughput screening methods are needed to efficiently identify these regulators.

Purpose of the Study:

  • To develop and validate an efficient, high-throughput method for identifying functional regulators of embryonic stem cell (ESC) renewal.
  • To pinpoint factors critical for ESC pluripotency and expansion using a combined screening approach.

Main Methods:

  • Utilized high-throughput short-hairpin RNA (shRNA) lentivirus screening in ESCs.
  • Employed concurrent measurement of cell number (alamarBlue assay) and alkaline phosphatase (ALP) activity in the same wells.
  • Incorporated secondary screening using immunofluorescence for morphological and marker analysis.

Main Results:

  • Demonstrated that concurrent alamarBlue and ALP assays efficiently identify multiple positive and negative regulators of ESC renewal.
  • Showcased the utility of shRNA libraries for persistent and high-efficiency gene down-regulation.
  • Validated the identification of key factors influencing ESC pluripotency and expansion.

Conclusions:

  • The described shRNA screening method provides an efficient and economical approach to identify multiple regulators of ESC renewal.
  • This technique is valuable for dissecting pathways governing ESC pluripotency and self-renewal.
  • The combined assay approach accelerates the discovery of critical factors in stem cell biology.