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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Tools for Improved Crop Biotechnology

Location: Crop Improvement & Utilization Research

Title: Gene targeting in embryonic stem cells, II: conditional technologies

Authors
item Ledbetter, Daniel -
item Thomson, James
item Piedrahita, Jorge -
item Rucker, Edmund -

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: April 2, 2014
Publication Date: N/A

Interpretive Summary: This article summarizes the impact that genome modification via transgenesis has had on the field of embryonic stem (ES) cells. The chapter discusses targeting by homologous recombination, the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), transposase (PiggyBac) and site-specific recombination-based technologies, and how these methods have changed genomic engineering. These technologies enables the removal of extraneous DNA and the gain-of function in a ubiquitous or tissue specific manner from the genome. From an application point of view, the paper discusses the type of specific genome editing technologies available along with the innovative strategies being developed.

Technical Abstract: Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombination has led to the development of exquisite models that have planned gene modifications fine-tuned down to the single base pair. In addition to gene targeting by homologous recombination, the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or PiggyBac transposase has gained considerable traction in the genetic engineering field to edit the mouse genome. In gain of-function studies, a variety of promoters have been used to drive transgene expression in a developmental-, tissue-, or cell-specific fashion. With the development of inducible systems (tetracycline and lactose based), it is also feasible to direct transgene expression in a time-dependent fashion. To circumvent the problems of secondary effects and embryonic lethality that arise from knockout mouse models (loss of function), the implementation of spatial and temporal deletion strategies has been both warranted and necessary. ES cells can be engineered with various recombination systems to generate conditional alleles for gene deletions, knock-ins, inversions, recombinase-mediated cassette exchange (RMCE), and chromosomal rearrangements. As of October 2012, ES cell clones with conditional alleles for 8897 genes have been generated.

Last Modified: 9/20/2014
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