Looking at cell death in


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Sana03.11.2017
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Overview

  • Looking at cell death in inxs embryos via double staining

  • Analysis of inxs candidate genes using Quantitative Real time RTPCR

  • Future Directions

  • Miscellaneous



inxs briefly

  • Dominant modifier of the irreC-rst phenotype

  • Phenotypic expression observed in the retina, showing reduction in cell death

  • Alleles P2, B94, and 3

  • Referred to in the lab as A, B, and 1237

  • Question – do inxs embryos have a cell death phenotype?





Tunel Staining

  • Detection of Apoptosis via the fragmentation of DNA

  • TdT labels the blunt ends of DNA breaks with a (fluorescein)labeled dUTP

  • Can be detected using fluoremetric techniques, or immunohistochemical techniques (ie anti-fluorescein antibodies).



Tunel Staining



Optimization of Double- staining

  • LacZ antibodies from Molecular Probes used at a 1:500 concentration.

  • Anti-mouse Cy3 was used as a secondary antibody.

  • Hot citrix treatment not used

  • Both reactions were fluorometric in order to obtain both signals (that worked the best)

  • Stage 15/16 was chosen because clear midline Lac Z staining could be seen



Double Staining Results



Double Staining Results



inxs Candidate Region



Question

  • Can we detect mRNA expression differences between inxs and wild-type tissues using QRTPCR?





Quantitative RTPCR

  • Primers were first tested on embryo RNA

  • Intended to test for differential expression using mutant retina RNA, but stage development was not consistent

  • Used white pre-pupae as a consistent tissue stage for mutant vs OreR RNA

  • Looked for lower expression in mutant vs OreR RNA

  • Best primer sets were used to look for expression in retina RNA









QRTPCR result summary

  • CG32407 and CG32412 were the only genes to show differential expression in the white pp

  • Unfortunately there is no sequence difference between the mutant allele 1237 and OreR in those genes

  • Four new gene predictions in the found in the inxs candidate region from Apollo Genome browser

  • PI38359, PI47229, PI51087, and PI65757

  • These genes were tested in Retina – only PI65757 worked



QRTPCR result summary

  • CG5592, CG5568, CG32410, CG32408, CG10483, CG10486 primer sets never seemed to work

  • Redesigned CG5568, 10483, 32408, 32410 using 1237 sequence

  • 5568 gave a working product in embryo but not in retina – can be eliminated?

  • Caveat – mutation may not affect mRNA levels, negative results are not truly definitive







Future Directions

  • Further sequencing of mutant DNA in candidate region

  • Further analysis of newly predicted genes using QRTPCR

  • Collection of RNA from A/B retinas for Quantitative Real-time RTPCR

  • Refining the map location of inxs using p-element system and molecular markers



Future Directions








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