Programmed Cell Death

Reljić, Boris, Stroud, David A., Boris Reljić, David A. Stroud

Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.