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Cloning Novel Genes Responsible for Sex Determination in T. scripta using Suppression Subtractive Hybridization
Goals:
The ultimate goal is to elucidate the molecular mechanisms of sex determination by comparing gene expression patterns for male and female pathways during embryonic development of T. scripta, an animal with Temperature-dependent Sex Determination. Available clones of genes related to sex determination in T. scripta include: Sox9, WT1 (Spotilla), DMRT1 (Kettlewell), AMH, SF1 (Wibbels), aromatase, AR, and ER (Crews). Novel genes that are differentially expressed in developing embryos at male-producing temperatures and female-producing temperatures will be detected using Suppression-subtractive hybridization (SSH) and differential screening. Putative novel genes will be confirmed by "Virtual Northern"analysis and sequence data homology searches in GeneBank.
Experimental Design:
Freshly laid eggs from the red-eared slider turtle, Trachemys scripta, were obtained commercialy (Robert Kliebert, Hammond, LA, USA). After transport to the laboratory, they were held at room temperature until we established embryo viability by candling of eggs. The eggs were randomized to eliminate any clutch effect, then placed in groups of 30 in covered trays containing moistened vermiculite. Egg trays were placed in reach-in incubators programmed to maintain a constant temperature at 26.0 or 31.0 ƒ C.
Tissue Collection and Total RNA Isolation:
One hundred and fifty adrenal-kidney-gonad complexes (AKG) were excised from developing embryos at each temperature for stages 14 through 19. Ninety AKGs were excised from embryos at each temperature for stages 21 and 23. Tissue was incubated in RNAlater at 4ƒ C overnight. The RNAlater was removed, and tissue was stored at -80ƒ C.
One hundred (~1 g of tissue) AKGs from each stage 14 temperature (26 and 31ƒ C) will be used for the first subtraction.
Frozen tissue will be ground under liquid nitrogen in mortar and pestel, homogenized with ice cold guanidinium thicyanate solution. Total RNA will be extracted with acid phenol chloroform treatment and purified by ethanol precipitation.
UV spectrophotometric determination of quality and quantity of RNA will be performed and integrity of the isolation will be checked by denaturing agarose gel electrophoresis.
Isolating mRNA from Total RNA:
mRNA will be isolated from total RNA using Promega’s PolyATtract mRNA Isolation system. Quantifictaion and purity ratio of mRNA will be determined by UV Spectroscopy. Store at –80 ƒ C until ready to use.
Desired yield for the isolation of the mRNA fraction of total RNA for each 26 and 31ƒ C set of animals is ~15-25 m g.
Northern Analysis of mRNA isolation:
Isolated mRNA samples will be run on a 1% denaturing agarose gel. RNAs will be blotted onto membrane and probed with 32P-labeled SF-1 and b actin.
Sense and antisense riboprobes will be made by in vitro transcription prior to RNA electrophoresis. Integrity of the probes will be confirmed by 6% polyacrylamide gel electrophoresis and subsequent development on X-ray film.
Expected results from Northern analysis for both the 26 and 31ƒ C preps should show a band corresponding to the size of the SF-1 transcript (5.8 Kb). b actin band should be between 1.35 – 2.37 kb.
The membrane will be stripped for storage and future use using Ambion’s Strip EZ.
Create 26ƒ C-specific and 31ƒ C -Specific Subtracted cDNA libraries using Subtractive Hybridization approach:
The PCR-select cDNA Subtraction Kit (Clontech) is a multi-step process with built-in checks and balances.
cDNA synthesis: Tester and Driver ds cDNA are prepared from the two mRNA samples under comparison using cDNA synthesis.
Rsa I digestion: Tester and driver cDNA are separately digested to obtain shorter, blunt-ended molecules.
Gel electrophoresis is used to analyze cDNA synthesis before digestion and to examine the efficiency digestion.
Adaptor ligation: Two tester populations are created with different adaptors, but driver cDNA has no adaptors. (Reactions will be prepared to run Forward-subtraction, Reverse-subtraction and Control-subtraction.)
Forward Subtraction enriches subtracted library for genes differentially expressed at 26ƒ C.
Reverse Subtraction enriches subtracted library for genes differentially expressed at 31ƒ C.
Ligation efficiency analysis will be performed through a PCR experiment which is designed to amplify fragments that span the adaptor/cDNA junction of each Tester set.
First Hybridization: Hybridization kinetics lead to equalization and enrichment of differentially expressed sequences.
Second Hybridization: Templates for PCR amplification are generated from differentially expressed sequences.
First PCR amplification: Using suppression PCR, only differentially expressed sequences are amplified exponentially.
An aliquot from each PCR reaction will be analyzed by agarose gel electrophoresis. A cDNA smear is expected from .2 – 2 kb.
Second PCR amplification: Background is reduced and differentially expressed sequences are further enriched.
Again, an aliquot from each PCR reactions will be analyzed by agarose gel electrophoresis. Expect to see a reduction in house-keeping genes (less smear, more distinct bands) in the secondary PCR reaction of the kit’s control skeletal muscle cDNA.
PCR analysis of subtraction efficiency: Comparison of the abundance of known cDNAs before and after subtraction. This is done with both a non-differentially expressed gene (housekeeping---ACTIN) and with a gene known to be differentially expressed between the two sources of RNA being compared.
Hybridization Analysis of Subtraction efficiency: Dot or Southern blot analysis of subtracted and unsubtracted secondary PCR products using different genes as probes is used to determine success of subtraction.
Cloning and Differential Screening of Subtracted Libraries:
T/A Cloning of Amplified Subtraction Products: Secondary PCR products from the Forward Subtraction (26 ƒ C AKGs) and Reverse Subtraction (31 ƒ C AKGs) will be cloned into pCR 2.1 vector from Invitrogen. Increased transformation efficiency will be achieved by electroporating into electrocompetent DH5a E. coli.
cDNA Array of Subtracted Clones: Random clones will be picked from Forward Subtracted cDNA library, plasmids isolated, and inserts amplified by PCR in 96 well plate format. PCR products will be purified and analyzed by agarose gel electrophoresis. PCR products will be used to prepare slot blots.
Random Primer Labelling of cDNA Probes: Forward-Subtracted, Reverse-Subtracted, Forward Unsubtracted and Reverse-Unsubtracted probes will be generated by in vitro transcription in the presence of a 32P-labelled dUTP using Ambion’s Stip EZ kit. Probes will be analyzed by polyacrylamide gel electrophoresis and specific activity determined by scintillation counts.
Hybridization with the Subtracted cDNA: Subtracted clones arrayed on nylon membranes will be hybridized with radiolabelled probes, washed, and exposed to x-ray film. The membranes will be stripped for reuse.
Confirmation of Putative Differentially Expressed Clones by Northern and Sequence Data Analysis:
Northern Analysis of Putative Differentially Expressed Clones: Clones identified as differentially expressed will be used to probe Northern blots of both 26 ƒ C and 31 ƒ C stage 14 Poly A+ RNA. Expected results should confirm that subtracted clones from the male-producing temperature are not expressed or expressed at a significantly lower level in the female-producing temperature. Additionally, size estimates of transcripts will be ascertained.
Sequence Data Analysis: Identification of novel clones will be achieved by sequence data homology searches in GeneBank.
Cloning and Differential Screening of Subtracted cDNA Library
T. scripta SSH Plan
