Preparation and Use of shRNA for Knocking Down Specific Genes

Ahmad Jamal; Saima Usman; Muy-Teck Teh; Ahmad Waseem

doi:10.1007/7651_2024_515

Preparation and Use of shRNA for Knocking Down Specific Genes

Methods Mol Biol. 2024 Feb 28. doi: 10.1007/7651_2024_515. Online ahead of print.

Authors

Ahmad Jamal¹, Saima Usman¹, Muy-Teck Teh¹, Ahmad Waseem²

Affiliations

¹ Centre for Oral Immunobiology and Regenerative Medicine, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
² Centre for Oral Immunobiology and Regenerative Medicine, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK. a.waseem@qmul.ac.uk.

PMID: 38411888
DOI: 10.1007/7651_2024_515

Abstract

Short hairpin RNA (shRNA) is a technique used to silence gene expression stably in various cells. There are however several reported problems. First, the cloning of oligos can lead to ligation of multiple copies; second, premature termination of sequencing reaction during confirmation of hairpin template; third, microdeletions/substitutions in hairpin during cloning; and fourth, off target effects. In this chapter, we have described a retrovirus transduction-based protocol that can be used on cells in culture without encountering any of the reported issues. We have used this protocol to clone shRNA templates for at least 10 different genes and confirmed them by dideoxy sequencing. The knockdown of 75-90% for two mRNA expressing genes, CDH5 and keratin KRT80, and a long non-coding RNA, XIST, is presented here.

Keywords: Dideoxy sequencing; Gene silencing; Oligonucleotide cloning; Retroviruses; Transduction.