Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

Abstract

Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano

“…Despite the mentioned limitations, random integration of foreign DNA appears to be a straightforward and productive approach for generating transgenic lines in M . lignano and can be used as a basis for further development of more controlled transgenesis methods in this animal, including transposon-based 42 , integrase-based 43 , homology-based 44 , or CRISPR/Cas9-based 45 approaches.…”

A platform for efficient transgenesis inMacrostomum lignano, a flatworm model organism for stem cell research

“…From these experiments we established the HUB1 transgenic line with ubiquitous GFP expression ( Supplementary Fig. 2e), for which stable transgene transmission has been observed for over 50 generations (Marie-Orleach et al, 2014, 2016.…”

“…Nevertheless, for the downstream genetic analysis involving transgenic lines, several rounds of backcrossing to nonirradiated stock might be required to remove any introduced mutations, which is easily possible given that these worms are outcrossing and have a short generation time (Marie-Orleach et al, 2016. Despite the mentioned limitations, random integration of foreign DNA appears to be a straightforward and productive approach for generating transgenic lines in M. lignano and can be used as a basis for further development of more controlled transgenesis methods in this animal, including transposon-based (Ivics et al, 2009), integrase-based (Fogg et al, 2014), homology-based (Gerlai, 2016) or CRISPR/Cas9-based (Komor et al, 2016) approaches.…”

Comprehensive behavioral analysis of indoleamine 2,3‐dioxygenase knockout mice

“…Too et al used Ido1 KO mice derived from 129/SvJ ES cells mated with B6 or CBA mice without a sufficient number of backcrossings, whereas we used Ido1 KO mice derived from 129/SvJ ES cells mated with B6 females and backcrossed with more than 10 generations of B6 mice. Genetic background may have a profound influence on behavioral phenotypes, and flanking genes might be responsible for the different phenotypes observed in mutant mice …”