Abstract
We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.
Highlights
We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds
Our proof-of-principle for AClashOfStrings vector concept (ACOS) and AGameOfClones vector concept (AGOC)/ACOS relied on the red flour beetle Tribolium castaneum, an emerging insect model organism[14,15], and covered the systematic creation of single and double homozygous transgenic lines
The subsequent mating procedure for the systematic creation of single homozygous transgenic lines spanned four generations (Figure S2) and was successfully completed for all three sublines, which eventually resulted in F7 and homozygotes (Table S1)
Summary
We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. Far more than four descendants must be produced and maintained in order to minimize the probability having no suitable individual available at the time of experimentation This economic as well as ethical issue can be addressed by establishing and applying deterministic workflows in which the genotype is identified directly upon nascence and production is stopped immediately once the number of suitable individuals is met. We already established the AGameOfClones vector concept (AGOC), which provides full visual control over one transgene and allows the systematic creation of homozygous transgenic lines[6] It uses mOrangebased[7] and mCherry-based[8] eye-specific[9] transformation markers (mO and mC, respectively), which are clearly distinguishable. Similar to AGOC, ACOS provides full visual control over one transgene, while the combined AGameOfClones/AClashOfStrings vector concept (AGOC/ACOS) provides control over two distinct transgenes
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