Ex#1MicroscopyProgeriaCells-01C - Exercise #1 Examination...

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Exercise #1Examination of Nuclear Morphology in Cells derived fromHutchinson-Gilford Progeria Syndrome (HGPS) Individuals andComparison to Cells from their ParentsIntroductionAs you have learned and read, Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease.  Individuals with HGPS die before the age of 20 years old due to symptoms of “old age” particularly cardiovascular problems and heart failure.  HGPS is caused by mutations in the lamin A (LMNA) gene.  Lamin A is a major component of the nuclear lamina.  Oneof the consequence of the LMNA mutation are defects in nuclear morphology.  In this study you willexamine the morphology of nuclei in cells from an individual with HGPS and also the cells from the mother and father of that individual for comparison and quantification.First, the fibroblastic cells derived from an individual with HGPS (termed the proband) willbe stained with Geimsa and the nuclei examined using bright field (light) microscopy.And, as agroup you will examine the nuclei of fibroblasts from the mother and father of the proband.Thenumber of “normal” and “deformed” nuclei in approximately 100 cells will be quantified in theproband, mother, and father.These data will be posted for the entire class and analyzed.These area few of the experimental questions that will be addressed in this study:1.Are all the proband cell nuclei deformed?2.What percent of the proband, mother, father cell nuclei are deformed?3.Do the mother and father have deformed nuclei?What are the percent deformed?Next, you will stain some of the cells with a fluorescent DNA binding dye (Hoechst).Wewill then visualize the nuclei by the confocal microscopy to obtain a different image of the nuclei,comparing the proband to either the mother or the father nuclei.Some of the general questions that you should be prepared to address in your lab report:1.Provide an explanation for the difference in percent deformed nuclei between the probandand the mother and father.2.Give a molecular explanation for the nuclear defects you visualized based on yourunderstanding of HGPS.3.Explain why the mother and father have some deformed nuclei?Overview of Procedures for this Lab:A.Practice using a Nikon light microscopeB.Stain Proband, mother, and father cells with Geimsa – View by light microscopy, count numbersof normal and deformed nucleiC.Stain Proband, mother and father cells with Hoechst (fluorescent DNA stain) – view nuclei byconfocal micrscopy1
Background Information – Microscopy and Cell Fixation and StainingThis study will also familiarize you with the use of bright field and confocal microscopy andsome basic methods used to prepare cells for microscopy.Microscopic examination of cells under avariety of conditions is a fundamental technique used in the investigation of cell structure andfunction.(Complete Online Assignment #1)There are now numerous optical methods that allow you to view cells, including bright field,

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Term
Spring
Professor
N/A
Tags
cells, bright field, B Stain Proband

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