WormBase Tree Display for Construct: WBCnstr00000352
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| WBCnstr00000352 | Summary | [myo-3::unc-68] | |
|---|---|---|---|
| Driven_by_gene | WBGene00003515 | ||
| Gene | WBGene00006801 | ||
| Construction_summary | A plasmid containing either the rab-3 or myo-3 promoter fused to the initial 1059 bp sequence of unc-68 was coinjected with cosmid M04C11. Cosmid M04C11 contains all the exons, introns, and 3' untranslated region (UTR) of the unc-68 gene. However, it lacks essential 5' upstream sequences for muscle cell expression and is unable to rescue unc-68 mutant phenotypes when injected alone. Homologous recombination was expected to occur in vivo between the plasmid and the cosmid, resulting in neuron- or muscle-specific expression of the transgene. The 1059 bp fragment of unc-68 was amplified from M04C11 by PCR using the following primer pair: ATA CCG GTC GAA CAA AAA TTA ATT TCT GAA GAA GAT TTA ATG GCC GAC AAG GAG GAG CA (sense) and CGA GAC CGC GGC TGA CCA TCT TCT ACG CCA TAC A (antisense). The sense primer included an AgeI site near the 5' end and a myc coding sequence fused in-frame to the unc-68 initiation site. The antisense primer had an SacII site near its 5' end. The PCR fragment was subcloned into two plasmids that contained either the rab-3 promoter plus rim 3'-UTR or myo-3 promoter plus the unc-54 3' UTR to form two new plasmids: wp137 for neuron-specific expression and wp235 for muscle-specific expression. Before injection, the cosmid and the plasmid were linearized with AgeI and SacII, respectively. AgeI does not cut the unc-68 gene. SacII cuts wp137 and wp235 only at the 3' end of the 1059 bp insert. M04C11 (13.2 ng/l), wp137 or wp235 (6.6 ng/l), and pPD118.20 (10 ng/l) were coinjected into unc-68(r1162) mutant animals for germ line transformation. pPD118.20 drives the expression of green fluorescent protein in body wall muscle cells and thus served as a marker of transformation. Successful recombinations between the plasmids and the cosmid were confirmed by PCR using a sense primer that is complementary to either the rab-3 or myo-3 promoter and an antisense primer that is complementary to the unc-68 gene downstream of the 1059 bp PCR fragment. | ||
| Used_for | Transgene_construct | WBTransgene00000352 | |
| Reference | WBPaper00026679 |
