The targeting vector (pHZ025) used to generate the krSi3(II) line was built as follows. A plasmid containing the unc-36
cDNA was provided by CI Bargmann (Saheki and Bargmann, 2009). This plasmid was digested by KpnI and EcoRI to remove egfp from the UNC-36 C-terminus, treated with T4 polymerase and circularized by self-ligation, resulting in the plasmid pHZ001. Next, egfp was fused in frame after the signal-peptide coding sequence in the unc-36
cDNA by PCR fusion. First, egfp was amplified using oHZ07 5'-TGGCCAGCTAGCAAAGGAGAA-3' and oHZ019 5'-TTCTTTTA TGCTCTCCTTATTAAAGTCCATGCCATGTGTAATCCC-3'. Next, the unc-36
cDNA was amplified using oHZ021 5'-ATGGCATGGACTTTAATAAGGA-3' and oHZ08 5'-AAAAGATCTTTTGTTTCTTCACGATTT-3'. Both fragments were then fused by PCR fusion using oHZ07 and oHZ08 and theresulting fragment was inserted into a MscI/BglII-digested pHZ001. A 2-kb region upstream of the unc-36
open reading frame was amplified from genomic DNA using oHZ09 5'-AAAAAAAAGCGGCCGCAAAATGCATGCAGTAATTAGTGTCC-3' and oHZ010 5'-TGGCCACTTATTAAAACTGCTTGT-3', and this sequence was inserted into the previous plasmid after NotI and MscI digestion, resulting in plasmid pHZ009. Finally the transgene sequence (Punc-36::egfp::unc-36
) was released from pHZ009 using NotI and ApaI, blunted using T4 Polymerase and inserted into the pCFJ151 vector backbone, resulting in pHZ025.