A Gene Vector that Facilitates Creation of Homozygotes

Nathaniel Grubbs, Ph.D.

Nathaniel Grubbs, Ph.D., is a regular contributor to Technology Topics. He is a postdoctoral research in the Department of Entomology, North Carolina State University MORE ABOUT THE AUTHOR

Recently, Stroble et al. (2018) published in the journal, eLIFE, a novel technique to quickly and efficiently develop homozygous transgenic lines. Although this method was tested in the genetic model organism, Tribolium castaneum, it could be of particular interest for entomologists seeking to develop transgenic tools in non-model organisms.

The technique, which they termed AGameOfClones (AGOC), employs a donor (transposon) that carries two marker genes (for their proof-of-principle, they used two different fluorochromes driven by the universal eye promoter, 3xP3). The two marker genes are each flanked with one copy of LoxN and one copy of LoxP, incompatible recognition sequences for Cre recombinase, in such a way that, in the presence of the recombinase, one of the two marker genes would get recombined out. The AGOC crossing scheme proceeded as follows:

  1. F0 (injectee) individuals were backcrossed to uninjected (WT) mates to obtain transgenic offspring (F1s), which were, once again crossed to WT mates. The backcross was repeated with the F2s to confirm that there is only a single insertion event.
  2. If F3s were indeed proven to have only a single insertion, they were crossed to individuals from a line which expresses Cre recombinase.
  3. The F4s were then backcrossed to the WT line to generate progeny (F5s) which only expressed one of the two marker genes.
  4. To ensure that homozygotes were generated, hemizygous F5s were crossed to siblings expressing the opposite marker. Then, heterozygous F6 siblings were crossed to each other to generate homozygotes expressing only a single marker.

The Cre/Lox site specific recombination system can be used to create deletions, inversion and translocations. This image is from a good overview of site specific recombination systems found on the Jackson Laboratory web site – here.

While this method may not be as quick as using balancer chromosomes, or establishing a homozygous line when the difference between hemizygotes and homozygotes is clear, balancers are not always available (certainly not in non-model organisms), and the differences between homozygotes and hemizygotes are not always obvious.

The 3xp3 promoter is popular because it can be used in a wide variety of insect species, resulting in marker gene expression in the optic lobes, ventral nerve cord and occasionally other neural cell types. While some have reported being able to distinguish between 3xp3 homozygotes from heterozygots, this is often not the case and establishing homozygous lines containing markers driven by 3xp3 becomes challenging in the absence of balancer chromosomes. This image is from Horn et al 2002

The authors describe several ways that AGOC has advantages over the use of genotyping to establish homozygous lines, and also suggest that this method might be advantageous when maintaining an insertion that is homozygous-lethal.

Finally, they offer some examples of why they think AGOC can be broadly applicable, as well as some tips to overcome a few likely pitfalls.

For researchers working with non-model organisms, or even those hoping to make their model work more efficiently, this paper is definitely worth a read. And for those that enjoy cool videos of embryonic development, check out the live images the authors took of some of the AGOC lines they generated during this work.

A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines. 2018 Frederic Strobl, Anita Anderl, Ernst HK Stelzer.  eLife 2018;7:e31677 doi: 10.7554/eLife.31677



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