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«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»

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RNA extraction RNA was extracted from Tribolium or Achaearanea embryos for synthesis of cDNA using acid guanidinium-thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987; Chomczynski and Sacchi, 2006) (Ambion, TRI reagent, A9738). Care was taken to avoid contamination of samples with RNases. Surfaces were wiped with RNasezap (AM9780). Quality of the extracted RNA was determined by running samples in denaturing 1% agarose gel in 1x TAE containing guanidium thiocyanate (Goda and Minton, 1995). RNA concentration and purity was determined by analysing samples with a Nanodrop spectrophotometer (Invitrogen, ND-1000).

cDNA synthesis cDNA synthesis was performed by reverse transcribing RNA using the RETROscript kit (Ambion, AM1710). A minus RT control was performed by using RNA template (without reverse transcription) to check for genomic DNA contamination.

Purified RNA that was contaminated with genomic DNA would result amplification of genomic DNA sequences that could include introns.

Primer design Sequences were obtained by RT-PCR for Tribolium. Sequences were obtained by RT-PCR for Achaearanea where sequence data was available. In order to obtain longer length sequences from small fragments of available sequence data for Achaearanea, RACE was performed.

A 1000bp clone of the Tribolium homologue of Dachshund (Tc dac) was given courtesy of Dr. Nikola-Michael Prpic-Schäper.

Primer sequences Primer sequences were designed manually using AmpliFX and computationally using primerBLAST and primer3 software. Primers were ordered from Eurofins MWG operon.

Degenerate PCR of At cnc Nested degenerate PCR conditions used for both inner and outer PCRs were as follows: initial hot start 94oC 5 minutes, denaturation step 94 oC 30 seconds, annealing step 45oC 45 seconds, elongation step, Final elongation 10 minutes. The PCR cycle was repeated 35 times.

The following products were obtained from combinations of the Inner/nested primers: KVAAQN and KYE/DLTE2 yielded a 71bp product (using SRDEKRA2 and KVAAQN, SRKEKRA2 KRKLDQI1, and KYE/DLTE2 and KRKLDQI1 as outer primer pairs).

KVAAQN and PIDEFNE yielded a 110bp product (using SRDEKRA2 and KRKLDQI1 as an outer primer pair).

Polymerase Chain Reaction (PCR) PCR reactions were performed in an thermocycler (Applied Biosystems, GeneAmp PCR system 2700). PCRs were performed in 50µl reaction volumes. The final concentration of reagents used for the majority of PCRs was as follows: dNTP 0.2mM, Magnesium Chloride 1.5mM, primer concentrations 0.4µM, taq polymerase (Applied biosystems, Amplitaq gold, 2 Units/reaction.

The basic cycling parameters used were as follows: Denaturation step at 94 oC 5 minutes, followed by a cycle of denaturation at 94 oC, annealing at various temperatures and an elongation step at 72 oC. This cycle was repeated 35 times followed by a final elongation cycle of 7 minutes. Annealing temperatures were used initially that were two degrees centigrade below the predicted T M (calculated using AmpliFX). PCR was optimised by varying annealing temperature if the initial PCR did not succeed.

PCR products were detected by gel electrophoresis by running through a 0.7%agarose gel in 1xTAE with 0.5µg/ml Ethidium Bromide at 4-7V/cm. PCR reactions that produced multiple bands were gel purified by cutting out the band on a UV transilluminator (UVP, model M-20) and extracting the DNA using QIAquick PCR purification kit (Qiagen, 28104) following the manufacturers’ instructions. PCR products that produced discreet products were precipitated with 1:10 PCR volume (usually 5µl) 3M Sodium Acetate pH5.2 (Sigma, S-7899) and two times the PCR volume (usually 100µl) of 100% ethanol. DNA was pelleted by centrifuging for one hour at 13,000g. The pellet was washed with 70% (v/v water) ethanol and air dried. The pellet was re-suspended in 20µl distilled water.

Cloning of PCR products PCR products (within one day of amplification) were ligated into pCRII (Invitrogen, K-2070) or pGEM T-easy (Promega, A1360) plasmid vectors by incubating overnight with T4 DNA ligase at 4 oC. Ligation reagents were present in the kit provided with the plasmid vector. TOP10 strain of E.coli cells (Invitrogen, C-4040) were transformed with ligated plasmids by heat shocking at 42 oC for 30 seconds.

Transformants were checked by blue/white screening and performing colony PCR of white colonies. PCR products were then analysed by gel electrophoresis. Colony PCR was performed by inoculating a PCR reaction mixture with one white colony. Primers used were M13 forward and reverse and the annealing temperature was 60 oC. The PCR products were run on a 0.8-2% agarose gel to check for the presence of an insert of the correct size. Picked transformant clones that were positive for the presence of an insert of the correct size were cultured for one hour in S.O.C. medium and cultured overnight in Lysogeny Broth (LB) at 225rpm at 37oC in a (Thermoscientific MaxQ 4450 barnstead lab line). Bacterial cells were pelleted from LB media by spinning at 2,600g for 15 minutes with a centrifuge (Fisons, Centaur-2 MSE). Plasmid DNA was extracted by using a QIAprep spin Miniprep kit (Qiagen, 27106) and following the manufacturors instructions. Verification of cloned fragments was performed by dideoxy sequencing of products using T7 and SP6 primers. Sequencing was performed out of house by commercial organisations (Source Bioscience). Plasmids were stored at minus 20 oC.

Hapten labelled RNA probe synthesis Labelled RNA probes were transcribed from PCR products from plasmid DNA that contained SP6 and T7 transcription start sites. PCR was performed using T7 and Sp6 primers or M13 forward and reverse primers.

A small aliquot of the PCR was analysed by gel electrophoresis by running through a 0.7%-2% agarose gel in 1xTAE with 0.5µg/ml Ethidium Bromide at 4-7V/cm.

The PCR products were purified by precipitating with 5µl 3M sodium acetate and 100µl ethanol at minus 20oC for at least one hour. The precipitated DNA was pelleted by spinning at 13,000g at 4oC for 30 minutes. The pellet was washed with 70% ethanol (in water). The DNA pellet was resuspended with 20µl water. The concentration and purity was determined using a spectrophotometer (Thermoscientific, Nanodrop 2000C).

Three haptens were used to label RNA probes. Digoxygenin (DIG), Fluorescein (FITC) and DNP. Antisense orientation of the cloned gene fragment was determined by DNA sequencing. 300-500ng of the purified DNA was added to a transcription reaction containing transcription buffer, RNase inhibitor. Purified PCR products were transcribed with T7 polymerase (Roche, 10881775001) or SP6 RNA polymerase (Roche, 10810274001) for four hours at 37oC in a water bath. DIG labelled RNA probes were labelled using DIG labelling mix (Roche 11277073910). FITC labelled RNA probes were labelled using Fluorescein labelling mix (Roche, 11685619910). DNP labelled probes were labelled by using a 10x Dinitrophenyl labelling mix (see Solutions and Media below). The transcription reaction mixture was incubated for 4 hours at 37 oC. 5µl water was added to the reaction, 1µl was then removed to be analysed on a 0.9% denaturing agarose gel by gel electrophoresis. The labelled RNA probe was hydrolysed by adding 25µl carbonate buffer and incubating at 65oC for 5 minutes. The hydrolysis reaction was stopped by adding 50µl stop solution. The labelled RNA probe was precipitated by adding 10µl 4M lithium chloride, 5µl torula yeast RNA and 300µl 100% ethanol. The labelled RNA probe was pelleted by centrifuging at 13,000g for 30 minutes and then washing the pellet with 70% ethanol. The probe was air dried for 5 minutes and resuspended in 200µl hybridization solution and stored at minus 20oC.

Synthesis of double stranded RNA dsRNA was transcribed from a PCR template which was flanked by two T7 promoter sequences. These flanker sequences were incorporated by performing PCR with an T7 promoter sequence primer together with an SP6 primer with a T7 promoter attached (T7-SP6 primer). Thereby adding a T7 sequence to one end of the PCR product. The PCR amplification step and purification was the same as for probe synthesis. The PCR was checked by gel electrophoresis and spectrophotometry for correct size, concentration and purity. 500µg-1000µg of PCR product was added to the T7 transcription reaction (Ambion, AM1334, MegaSCRIPT T7 kit). The reaction was performed at 37oC for 4 hours. The dsRNA was purified by lithium chloride precipitation. 30µl 7M lithium chloride and 30µl nuclease free water was added to the transcription reaction and incubated at -20oC for one hour. The dsRNA was pelleted by spinning at 13,000g for one hour. The pellet was washed with 70% ethanol and air dried before being resuspended with 20µl water. A 1µl aliquot was analysed by spectroscopy to check concentration and purity. The dsRNA was denatured and reannealed by heating at 90oC for 5 minutes then letting the hot plate cool to 70 oC (about 15 minutes). The dsRNA was stored at minus 80oC prior to injection.

Degenerate PCR Most sequences of cnc orthologues were retrieved from the Nucleotide database (http://www.ncbi.nlm.nih.gov/nucleotide/).Ixodes scapularis cnc orthologue was retrieved from Vectorbase (http://www.vectorbase.org/). Sequences were aligned with the alignment program Maclade and ClustalW. Numerous sets of primers were designed against conserved sequences.

Rapid amplification of cDNA ends (RACE) Both 5’RACE and 3’RACE were performed using First choice RLM-RACE kit (Ambion, AM1700) according to the manufacturer’s instructions. Nested PCR was performed to increase specificity of the PCR reaction.

8.3 in situ hybridization protocols.

Tribolium Embryo collection To collect embryos for fixation, adult beetles were placed on 300µm preseived organic white flour supplemented with 5% brewer’s yeast at 32 oC. Embryos were sifted from white flour with a 150µm sieve. Germ band extending embryos were obtained 0-24 hours after egg laying on white flour. Germ band retracting embryos were obtained 24-48 hours after egg lay. Very late stage embryos (undergoing dorsal closure) were obtained 48 hours after egg lay.

Fixation of Tribolium embryos.

Embryos were dechorionated by immersing in 25% (v/v) bleach and 75% (v/v) distilled water for 5 minutes. Embryos were rinsed in wash buffer and distilled water.

Embryos were fixed in fixation buffer with 9.25% formaldehyde for thirty minutes at room temperature at 225rpm on a table top shaker. Embryos were devitellinized by methanol shocking. Fixation buffer was removed prior to adding 100% methanol to the vial. The vial was shaken vigorously for thirty seconds and any embryos that sink to the bottom are collected. Remaining embryos, particularly late stage embryos, were devitellinised by violent aspiration with a 0.9mm syringe. This was repeated several times. Embryos were stored at -20oC for at least one week before being used for in situ hybridization experiments.

Tribolium enzymatic whole mount in situ hybridization of Tribolium embryos protocol The whole mount in situ hybridization protocol was adapted from a fluorescent in situ hybridization protocol (Kosman et al., 2004) and Tribolium in situ protocol (Schinko et al., 2009). Embryos were kept in 1.5ml microcentrifuge tubes. All washing steps and incubation steps were performed at room temperature on a rotatory wheel unless otherwise stated. Embryos were given two minutes at the end of every step to settle at the bottom of the eppendorf to minimize loss of germ bands.

Embryos were transferred from methanol to PBS-T and then post-fixed in 4.5% formaldehyde. Proteinase K was added to the embryos at a concentration of 4µg/ml and incubated for 8 minutes. Embryos were rinsed several times over a period of thirty minutes with PBS-T in after the post-fixation and proteinase K digest steps. Postfixation was repeated at the same concentration. Embryos were washed several times over the period of an hour after the final post-fixation.

Embryos were transferred gradually to hybridization solution. Incubation steps in hybridization solution were performed at 60 oC in a water bath. The embryos were washed at least four times with pre-heated hybridization solution over a period of at least two hours. The washing steps were performed in a heating block at 60 oC to ensure constant temperature.

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