WormBase Tree Display for Transgene: WBTransgene00000421
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| WBTransgene00000421 | Summary | [phf-5::yfp] | |
|---|---|---|---|
| Synonym | EC610 | ||
| Construction | Construct | WBCnstr00000421 | |
| Integration_method | Particle_bombardment | ||
| Construction_summary | The C. elegans phf-5 gene (6.9kb) comprising all phf-5 coding and upstream sequences was amplified using gene-specific primers. Gene-specific forward and reverse primers (10 pmol primers RepC.e.F1: 50-TCCAGTAGCAGCAGTACCCCGCC-30 (located 2.8 kb upstream of the translation initiation codon in exon 1) and RepC.e.R1: 50-TCTTCGCTGCCTTGTCGACGC-30 (located 1.0 kb downstream of the stop codon in exon 3)) were used. 0.5 ul of a 1:100 dilution of this product was subjected to a second round of PCR using gene-specific primers with attached recognition sites for the endonucleases BamHI and XhoI, i.e., RepF1 (XhoI): 50-CGGCTCGAGTCCAGTAGCAGCAGTACCCCGCC-30 (located 2.8 kb upstream of the translation initiation codon in exon 1) and RepStop (BamHI): 50-CGGGATCCGACTTTTTCGATCCGAATTTCTTTCG-30 (located in exon 3: codon 102110). The resulting 5.8 kb amplicon was subsequently digested with the restriction endonucleases PstI and BamHI, resulting in a fragment of 5.5 kb comprising all phf-5 coding, intronic, and 2.5 kb of upstream sequences, which was subcloned into the PstI/BamHI sites of the pBluescript vector. Subsequently, a bombardment vector was createdby transferring the wild-type unc-119 gene of plasmid pMM016 as a HindIII/XbaI restriction fragment into the NheI/HindIII cut vector pEYFP-N1 (BD Clontech, Palo alto, CA), resulting in pEYFP-N1-unc-119. The C. elegans phf-5 gene was inserted and transcriptionally fused to the enhanced yellow fluorescent protein (EYFP) coding sequence by cloning the phf-5 gene from the pBluescript plasmid as a XhoI/BamHI restriction fragment into the SalI/BamHI cut pEYFP-N1-unc-119 construct, resulting in phf-5-EYFP-N1-unc-119, which was then used to transform C. elegans strain DP38 unc-119(ed3) III by particle bombardment.Two independent transgenic lines (EC610, EC611) were obtained as integrates into the C. elegans genome by particle bombardment of C. elegans strain DP38 unc-119(ed3) with the phf-5-EYFP-N1-unc-119 plasmid and gave identical pattern of EYFP expression. --precise ends | ||
| Used_for | Expr_pattern | Expr2000 | |
| Reference | WBPaper00005466 | ||
| Species | Caenorhabditis elegans |
