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Fig. 1 | BMC Genetics

Fig. 1

From: A toolset to study functions of Cytosolic non-specific dipeptidase 2 (CNDP2) using Drosophila as a model organism

Fig. 1

Experimental design for generation of null and rescue alleles of the dCNDP2 gene. a Schematic representation of the dCNDP2 locus and the donor plasmid pGX-5′&3′-dCNDP2-null, which contains the mini-white reporter gene (red) flanked by the left and right homology arms (brown). The gRNA target sites are indicated by vertical arrows. b Modifications introduced into the dCNDP2 locus. In step 1, the dCNDP2 gene was replaced by the mini-white reporter gene using CRISPR/Cas9-mediated HR approach. Note that the reporter gene is flanked by two loxP sites. In step 2, the mini-white reporter was removed by Cre recombinase-mediated recombination between loxP sites. This resulted in generation of a null allele (∆dCNDP2), in which the dCNDP2 gene is replaced by the attP and loxP sites. In step 3, the rescue construct pGE-attB-GMR-dCNDP2 was integrated into the attP site by phiC31 integrase-mediated recombination. The rescue construct contains the mini-white reporter gene, which upon integration into the genome becomes flanked by two loxP sites, and the genomic DNA fragment carrying the dCNDP2 gene that is absent in ∆dCNDP2 mutants. In step 4, the mini-white reporter was removed by Cre recombinase-mediated recombination between loxP sites. This resulted in generation of the ∆dCNDP2(rescue) transgenic flies, in which DNA sequence of the dCNDP2 locus is almost identical to the wild-type one except for several single nucleotide variations (see Methods) and the presence of the attR and loxP sites. Black horizontal arrows indicate primers used for PCR genotyping (for primers sequences, see Additional file 1: Table S1). Plasmids are drawn as circles with the relevant elements indicated; Ampr, ampicillin resistance gene; ori, plasmid origin of replication

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