Pcr topo 2

The primers were designed for amplification of specific regions for myoVaa (NM_001080959.2; bp 2880–3795), myosin Vb (NM_001161632.1; bp 2967–3866), myoVc (XM_686051.3; bp 2920–3720) using Primer3 (version 0.4.0) and cloned into pCR TOPO 2.1 or pCR TOPOII (Invitrogen). For RNA probe synthesis, templates were linearised and probes were synthesized using either T7 or SP6 RNA polymerase (Roche DIG RNA Labelling Kit). In situ hybridisations were performed as described [82] with a few modifications. For sectioning, embryos were post-fixed in 4% PFA after the completion of in situ hybridisation protocol and embedded in Epon. Sections were cut at 3 µ thickness using glass knives on Leica microtome, placed on a glass slide coated with 1% gelatin, counterstained with 4% eosin made in 90% ethanol and mounted in DPX. Sections were imaged on Zeiss ApoTome using Axiocam.

TRIzol-extracted RNA (10 μg) was resuspended in loading buffer (1× morpholinepropanesulfonic acid-EDTA-sodium acetate [MESA; Sigma], 4.5% formaldehyde, and 32% formamide), denatured for 15 min at 65°C, separated in a 1.2% agarose gel containing 5.92% (vol/vol) formaldehyde and 1× (vol/vol) MESA, and transferred via capillary action to a Zeta-probe membrane (Bio-Rad) overnight at room temperature. RNA was cross-linked to the membrane using a TL-2000 (UVP) and then stained with methylene blue to visualize transfer of RNA. Methylene blue was removed by washing the membranes in 1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–1% SDS for 15 min three times and then prehybridized in 5 ml ExpressHyb (ClonTech) for 1 h at 65°C. The hybridization buffer was changed, and a ³²P-labeled dsDNA probe (Invitrogen) was added for 1 h at 65°C. Blots were then washed with 0.1× SSC–0.1% SDS three times for 15 min at 55°C, exposed overnight to a phosphorimager screen, and subsequently visualized on a Typhoon 9400 (GE). The ZIKV 3ʹ UTR probe (targeting nucleotides 10324 to 10808 of the viral genome) was generated by reverse transcription-PCR amplification of the 3ʹ UTR from ZIKV-infected cells, and the resulting PCR product was cloned into pCR-TOPO2.1 (Invitrogen). The actin and ZIKV probes were randomly labeled with [α-³²P]dATP (Perkin Elmer) using a RadPrime labeling kit (Invitrogen).

Genomic DNA from the wild type strain AB was used to amplify bait sequences by PCR (Phusion Polymerase, Thermo Fischer) using primers listed in the Supplementary Table S2. To generate donor plasmids, baits were cloned into a pCS2+ or Topo PCR II vectors (Invitrogen) containing the coding sequence for the Venus fluorescent protein or turboRFP (promoter-less tRFP plasmid, Evrogen). The CMV promoter was later removed from the pCS2+ vector. All constructs were verified by sequencing.

To determine if trpm channels are expressed by skin melanophores we assessed mRNA expression by RT-PCR. Total RNA was obtained from whole embryos (positive control) and isolated tails (cut below the cloaca to separate the tail and upper body) using TRIzol (Invitrogen) according to the manufacturer’s protocol. Isolated tails were analyzed as a substitute for the skin, given the absence of organs and specialized tissues within the tails relative to the whole embryo. Single-strand cDNA was produced from RNA samples (5 µg) by priming with oligo(dT) primers using SuperScriptTM IV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. All PCR amplifications were carried out in a total volume of 20 µL with 1 µL of cDNA, 2 µL of primers, 7 µL of water and 10 µL of 2X PCR master mix (Thermo Scientific, IL). PCR amplifications were carried out at 45 cycles and with an annealing temperature of 55 °C. PCR products obtained from cDNA were cloned into TOPO-pCRII (Invitrogen) vectors and sequenced to confirm identity. The cloned sequences were submitted to GenBank-NCBI (Supplementary Table 1).

Eyes were fixed in modified Carnoy’s fixative, embedded in paraffin, and sectioned as described above. Riboprobes were generated from gene fragments using cDNA pooled from E7 anterior eyes and cloned into TOPO-PCRII (Invitrogen). Digoxigenin (DIG)-labeled riboprobes were synthesized following the manufacturer’s protocol (DIG Labeling Kit, Roche). Primers used to generate the riboprobes are listed in Supplementary file 1. Sections were hybridized with riboprobe at 52°C (Npnt) and 60°C (Itgα8) overnight. Hybridization was detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche) and color was developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma). Following color development, sections were fixed with 4% PFA, mounted in Cytoseal, and imaged using an Axiocam mounted on an AxioImager2 microscope (Carl Zeiss).

Total RNA from cavefish embryos at multiple stages was reverse transcribed with random primers using AMV reverse transcriptase (Promega). Sequence for Hcrt transcript was obtained from our own next-generation sequencing data, and partial 750 bp sequence was amplified by PCR using specific primers. PCR products were subcloned in TOPO-PCR II vector (Invitrogen, Carlsbad, CA, USA) and sequenced. They corresponded to XM_007287820.3 (Hcrt precursor mRNA predicted from the genome). Pomcb, Pomca, AgRP, IT, AVT, CART3 and NPY cDNAs were obtained from our in house clonal library of ESTs (accession numbers FO375681, FO257910, FO289826, FO221370, FO234678, FO230154, FO263072). PCR products were sequenced by Sanger method before probe synthesis. Lhx9 (NM001291259.1) and Lhx7 cDNA were previously cloned by our group and by David Stock, respectively. These two LIM-homeodomain factors were chosen for analysis because unpublished work in the group had mapped their expression domains in the exact territories where Hcrt and NPY cells (modified in numbers in cavefish) are located.