Genetic manipulation of mosquito vector competence of the type

Transformation vector For [SM1]4, a synthetic gene coding for four units of the SM1 peptide (PCQRAIFQSICN) separated by 4-amino-acid (GSPG) linkers was constructed as follows. Two oligonucleotides, SM1þ (5 0 -CCCGTGCCAGCGCGCCATCTTCCAGTCGATCTGCAA CGGCTCGCCGGG-3 0 ) and SM1 (5 0 -GCCCGGCGAGCCGTTGCAGATCGACTGGAA GATGGCGCGCTGGCACGG-3 0 ), were annealed, phosphorlylated and self-ligated. The ligation products were fractionated by gel electrophoresis and the 4-repeat unit was excised from the gel to yield [SM1]4. Two adaptors, 5 0 and 3 0 , were added to [SM1]4. The 5 0 adaptor was obtained by annealing 5 0 -CGGATCCCCGGG-3 0 and 5 0 -GCCCGGGGA TCCGGTAC-3 0 , and the 3 0 adaptor by annealing 5 0 -CTACCCCTACGACGTGCCCGAC TACGCCG-3 0 and 5 0 -GATCCGGCGTAGTCGGGCACGTCGTAGGGGTA-3 0 . The 3 0 adaptor codes for the HA1 influenza haemagglutinin epitope. For 5 Cp, a 1.8-kilobase (kb) KpnI–KpnI fragment containing the A. gambiae CP 1.7kb promoter, 5 0 untranslated region (UTR) and signal peptide down to nucleotide þ125 (ref. 9) was obtained by PCR with T7 (5 0 -GTAATACGACTCACTATAGGGC-3 0 ) and AgCPKpn (5 0 -GGTACCCTCGGCCGCTTCGACACT-3 0 ) primers using the pBluescript AgCP genomic subclone as a template, followed by digestion with KpnI. For 3 Cp, a 555-base pair (bp) fragment containing the CP 3 0 region (including the stop codon and 3 0 UTR; nucleotides þ1,337 to þ1,880) was obtained by PCR with the primers AgCP3BH (5 0 -GGATCCTGAAGTCTCTCCTACCGG-3 0 ) and AgCP3Sc (5 0 CCGCGGTAAGGCTAGCATTGCCA-3 0 ) using the AgCP pBluescript genomic subclone as a template, followed by digestion with BamHI and SacII. The three fragments, 5 0 Cp, [SM1]4 with adaptors, and 3 0 Cp, were combined and sub-cloned into pGEM-T Easy vector (Promega), then digested with NotI and inserted into the NotI site of pSLfa1180fa (ref. 11). This construct was digested with FseI and AscI, and inserted into the FseI–AscI site of pBac[3xP3-EGFPafm] plasmid to yield pBacAgCP[SM1]4.