A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines.

Frederic Strobl,Anita Anderl,Ernst Hk Stelzer

doi:10.7554/elife.31677

Abstract

Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.

Highlights

Life sciences, especially cell and developmental biology, rely on model organisms
The proof-of-principle of the AGOC vector concept relied on the red flour beetle Tribolium castaneum, an emerging insect model organism (Klingler, 2004; Brown et al, 2009), in conjunction with the piggyBac transposon system (Lorenzen et al, 2003; Berghammer et al, 2009), which allows semi-random genomic insertion
The unique feature of our approach is that temporary ambiguities are avoided in any generation, since all genotypes are directly identified by designed distinct phenotypes

Summary

Introduction

Especially cell and developmental biology, rely on model organisms. The most frequently used vertebrates are mouse and zebrafish. Only costly and error-prone methods can distinguish between these individuals To overcome these drawbacks, Strobl et al developed a concept called AGameOfClones and applied it to the red flour beetle Tribolium castaneum. Strobl et al developed a concept called AGameOfClones and applied it to the red flour beetle Tribolium castaneum In their approach, the transgene expresses two marker-proteins with different fluorescent colors. The AGameOfClones concept has several major advantages: individuals with different markers can be identified, the procedure is cost-efficient and reliable, and it can be applied to most model organisms. This will benefit breeding schemes and animal welfare since irrelevant individuals can be excluded as soon as the markers become detectable.

Results

Discussion

Materials and methods

Funding Funder