74, 176, 177, 178, 179, 180).Culex quinquefasciatus mosquitoes used in this study were from a laboratory colony maintained at UC Davis. This colony was initiated with adult mosquitoes from a colony maintained by A.J.C. at the Kearney Agricultural Center, University of California, andJ Insect Physiol. Author manuscript; available in PMC 2014 September 01.Xu et al.Pagestarted from mosquitoes collected in Merced, CA in the 1950s. In Davis, mosquitoes were kept in an insectary at 27 , under a photoperiod of 16:8 h (L:D) for the last 3 years.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript2.3. Cloning of OR genes from Cx. quinquefasciatus Total RNA was extracted from one thousand 1-day-old female Cx. quinquefasciatus antennae with TRIzol reagent (Invitrogen, Carlsbad, CA). Antennal cDNA was synthesized from 1 .. g of antennal total RNA using SMARTerTM RACE cDNA amplification kit according to manufacturer’s instructions (Clontech, Mountain View, CA). To clone their ORFs into pGEMHE vector, PCR was performed with the following gene specific primers with restriction endonuclease sites (nucleotides upstream of the restriction sites were omitted for brevity): CquiOR1 Fwd-XmaI (underlined) primer 5 2 CCCGGGATGAAATTCGCTCCGCTCCAG-3 2 Rev-XbaI (underlined) primer, 5 and 2 TCTAGATCAGATTCTTTCCTTCAGCAC -3 ; CquiOR44 Fwd-XmaI (underlined) primer, 2 5 -CCCGGGGGGAATGGACACCTGTGCGCATCAG-3 2 Rev-HindIII (underlined) 2 and primer, 5 -AAGCTTGGGTTATTTCGTCACCTCGAGCAG -3 ; CquiOR73 Fwd-XmaI 2 2 (underlined) primer, 5 -CCCGGGACCATGTCGTCCATCAACCTTCCAT-3 2 Rev2 and HindIII (underlined) primer, 5 -AAGCTTGCTCTAGA 2 TCATTCCTCTGCGTAGAGCTGTTG-3 ; CquiOR87 Fwd-XmaI (underlined) primer, 5 2 2 CCCGGGGGGAATGAATGACAGTTACAATGTTG-3 2 Rev-XbaI (underlined) and primer, 5 -TCTAGAGCCTACATTTTGCTCCCCATC-3 ; CquiOR110 Fwd (1)-XmaI 2 2 (underlined) primer, 5 -CCCGGGGGGAATGGGAATTACCTGTAGTTG-3 , Rev (1)-XbaI 2 2 (underlined) primer, 5 -TCTAGAGCTTACTCAAACACGCTGAG-3 ; CquiOR110 Fwd 2 2 (2)-XmaI (underlined) primer, 5 -CCCGGGGGGAATGGACTTGAGCTTCATGTTG -3 , 2 2 Rev (2)-XbaI (underlined) primer, 5 -TCTAGAGCTTAATGTCCCCACGGTAGAAC -3 ; 2 2 and CquiOR161 Fwd-XmaI (underlined) primer, 5 2 CCCGGGGATGGCCAACCGAAGAAAGCTC -3 2 Rev-HindIII (underlined) primer, and 5 -AAGCTTTTACATATTTTGCAACATCAT -3 .Daclizumab 2 2 PCR amplifications were performed using Pfu Ultra II polymerase (Stratagene, La Jolla, CA) under the following condition: 5 cycles of 94 for 30 s, 57 for 30 s, 72 for 3 min, and 30 cycles of 94 for 30 s, 55 for 30 s, 72 for 3 min, and then 72 for 10 min.Inavolisib PCR products were purified using QIAquick Gel Extraction kit (Qiagen, Valencia, CA), ligated into EcoRV site of pBluescript SK (+) (Stratagene) using T4 DNA ligase (Promega, Madison, WI) and transformed using One Shot TOP 10 competent cells (Invitrogen, Carlsbad, CA).PMID:23849184 After screening colonies, plasmids were extracted using the QIAprep Spin Miniprep kit (Qiagen) and sequenced by ABI 3730 automated DNA sequencer at Davis Sequencing (Davis, CA). Plasmids were digested with appropriate restriction enzymes (20 U/.. l) for 2 h at 37 . Digested products were purified using QIAquick Gel Extraction kit (Qiagen), ligated into pGEMHE, and transformed using One Shot TOP 10 competent cells (Invitrogen). Plasmids were extracted using the QIAprep Spin Miniprep kit (Qiagen) and sequenced by ABI 3730 automated DNA sequencer at Davis Sequencing (Davis, CA) for confirmation. 2.4. Quantitative analysis of OR gene expression (qPCR) Antennae fro.