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Figure 1 | BMC Biotechnology

Figure 1

From: Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method

Figure 1

Schematic overview of UV/TMP low-copy integration. (A) Positive selection marker (vps-45 mini gene), negative selection marker (ben-1 genome) and injection marker (P myo-2 ::venus) were co-injected into the recipient strain (tm234(ben-1);tm246(vps-45)) to generate transgenic strains carrying Ex arrays. Ex strains were treated by UV/TMP to obtain insertion (Is) strains. Is strains were cultured at 20°C on benzimidazole-containing NGM plates. Under these conditions, single- or low- copy integrated strains survived but multi-copy integrants or Ex array-carrying animals did not survive due to multi-copy transgenes containing the ben-1 gene. Strains were further tested by PCR#A and PCR#B to identify the intact floxed vps-45 mini gene. PCR-selected lines were backcrossed with N2 wild-type to remove vps-45 and ben-1 mutations, followed by microinjection of Cre recombinase mRNA to excise the vps-45 mini gene. Cre-LoxP excision was detected by PCR#1. qPCR1 and qPCR2 indicate regions amplified by quantitative PCR described below. (B) Relative amount of the vps-45 gene determined by quantitative PCR with primers that amplify the 5' region in the vps-45 mini gene (gray bars: qPCR1) and the middle region (black bars: qPCR2). The amount of the vps-45 gene (normalized to the act-2 gene) is presented as a ratio to the N2 control. Error bars represent SE of three independent experiments.

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