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Molecular Online Test 2 You are studying a new cancer oncogene called ORANGE CRUSH

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Molecular Online Test 2

You are studying a new cancer oncogene called ORANGE CRUSH. You have preliminary data of its DNA and Protein Sequence. It is a mutant of the protooncogene ORANGE, a GTPase binding protein related to RAS at about 50% amino acid identity. NOTE, The ORANGE CRUSH mutant gene is always in the GTP bound form (constitutively active). Assume you have all antibodies needed for all the proteins studied.

Questions 1, 2, and 3 worth 35 points, while #4 is worth 45 points. 

 

1) You need to develop cell lines of Transient and Stable nature for further study.

•             Using the material from the gene expression lectures, document the controls and experimental for making ORANGE CRUSH Transient and Stable cell lines.

•             Describe all relevant cancerous phenotypic analyses of these cell lines with controls.

•             Compare and contrast RAS cell lines vs. ORANGE CRUSH cell lines with controls.

 

2) You next design and use key gene microarrays to continue your study of Transient and Stable ORANGE CRUSH cell lines.

•             Describe and discuss the use of microarrays to discover data of gene expression in these cell lines.

•             Given the relatedness to RAS, use String looking at RAS to estimate essential partner proteins to test by protein partner analysis. Show and describe this process to find similar ORANGE CRUSH protein partners with controls.

•            

3) You next move to RNA Seq experiments to test various hypotheses concerning the ORANGE CRUSH oncogene.

•             Describe using RNA Seq analysis experiments with controls to figure the expression levels in the two cell lines you made, as well as an available biopsy from patients with ORANGE CRUSH cancer.

•             Use RNA seq to determine the level of the predicted protein partners found from question 2. As a check, use known RAS protein partners RNA Seq data in RAS cancer cell lines. (Pubmed?)

•            

4) Mutagenesis Analysis of ORANGE CRUSH

•             Using Mutagenesis experiments, change the other critical amino acids in ORANGE CRUSH using know RAS mutations as a guide.  Discuss confirmation experiments for making these other ORANGE CRUSH mutants

•             Express these NEW OC mutants in a stable cell line and run them through the phenotypic tests, Microarray Analysis, and RNA seq metric experiments with controls.

 

Bonus+10. If cell lines expressed both RAS and ORANGE CRUSH mutant genes, what are your predicted results with controls? Discuss and show confirmational experiments as well.