Biology

Name: ! BME 302 Cellular Engineering HW 2 – Cell Signaling & Assays (112 points) Due in class February 16, 2016 ! Section 1 – Cell Signaling Please provide concise and clear answers – WRITE WELL ! 1. What Kind of Signals Do Cells Receive? List and describe the main types (4) 2. What’s the difference between neurotransmitters and the follicle-stimulating hormone

signaling molecules? (4) 3. How Do Cells Recognize Signals? What types of cellular signal-receiver exist? (4) 4. How Do Cells Respond to Signals? (4) 5. What are the second messenger and give an example (4) 6. Why do protein conformational changes affect cell signaling? (4)

Sarah Al Ameer
Growth factors: factors that promoting growth and tissue repair. Hormones: Long range signal molecules to trigger action of target. Neurotransmitters: Short range signaling molecules that travel between neurons and muscle cells. Extracellular matrix components.

7. What’s the role of protein kinase and protein phosphatase and what is the mechanism of action? (4)

8. How Do Signals Affect Cell Function? (4) 9. Based on the wnt signaling pathways that is schematized below

what do you think will happen to the cell if you – knock-out IP3 (2) – overexpress CKIa (2) – knock-out NKD (2)

The Calcium ion channels will not open in the cell membrane, therefore cannot release Calcium ions in large amounts to activate protein kinase C.

10. Based on the TGF-beta signaling pathways that is schematized below

what do you think will happen to the cell if you – Overexpress Smad7 (2) – Knock-out Smad 2/3 (2) – Inhibit PTHR by using soluble receptor ligands (2)

11. What does CELL PHENOTYPE mean? (4) 12. What’s the difference between quantitative and qualitative assessment of cell phenotype.

Prove examples for both. (4) 13. You have cell A and cell B. Cell A changes phenotype after you place it in an in vitro co-

Precievable characteristics and traits of gene.

culture system with cell B. How do you determine if this is the result of paracrine or contract- dependent signals? (4)

14. You start a culture of stem cells and you use a protocol to differentiate them into beta cells.

To make sure they are truly beta cells. First you want to check whether the insulin gene is expressed at higher levels in the differentiated vs. the undifferentiated cells. What methods that we studies would you use and describe the step by step procedure. (4) Then you want to demonstrate that they secrete insulin when you place glucose in their culture media. What methods that we studies would you use and describe the step by step procedure. (4) Finally, you want to show that inside differentiated cells insulin is located within secretory vesicles and quantify in which percentage of the cells. What methods that we studies would you use and describe the step by step procedure. (4)

15. You hypothesize that a group of tolerogenic cells is located in the fetal pancreas. What method would you use to identify their location within the tissue? (4) What method would you use to quantify their abundance in the whole fetal pancreas based on their surface expression of protein A and Protein B and the absence of protein C? (4)

16. What is the difference between genomic DNA and cDNA? (4)

17. You need to determine the effects of the duration of treatment with ethylene vs.

ethylene+A23187 on calmodulin gene expression. You extract total mRNA from your cells, generate cDNA by reverse transcription and run end-point PCR. You run your PCR product on a gel and you get the following picture. Draw a bar plot to quantify the following results of end-point PCR. (4)

18. You need to determine the effects of knocking-down (KO) the CIZ gene on expression of the

following genes: COL1, ALP, OPN, OSX. You extract total mRNA from your control cells: wild type (WT) cells, and from your genetically engineered cells (CIX-KO), then you generate cDNA by reverse transcription and run end-point PCR. You run your PCR product on a gel and you get the following picture. Draw a bar plot to quantify the following results of end-point PCR. (4)

19. You need to quantify expression of the INS gene on several stem cell derived insulin-

secreting cell products (SCb) that you have generated: SCb1, SCb2, SCb3, SCb4, SCb5, SCb7 and choose the product that displays the highest INS expression. You extract total mRNA from all your seven cell products, then you generate cDNA by reverse transcription and run real time PCR and you get the following plot. (8)

Make a table with the Ct for each sample. (4) Assuming that all seven samples had a Ct of the endogenous gene of 16 and a Ct of the INS

gene for the control cells (islets) of 10, express the expression of the INS gene for each cell product relative to islets. (4)

20. If you run a real time PCR on your cDNA samples and you get the following plot, what does it

mean and what are the possible causes? (4)

21. You need to characterize the vascularization of an engineered pancreatic tissue (from mice).

You have prepared the tissue by fixing it in 10% formalin and by sectioning it into 5µm-thick sections. Now you decide to perform in situ immunohistochemistry. (4) You have the following primary antibodies: Rabbit anti-mouse CD31 (will stain all endothelial cells in blood and lymphatic vessels) Goat anti-mouse Lyve-1 (will stain only endothelial cells in lymphatic vessels) Guinea pig anti-mouse INSULIN (will stain all beta cells) Rabbit anti-mouse GLUCAGON (will stain all alpha cells)

And these secondary antibodies: Chicken anti-rabbit HRP Chicken anti-rabbit AF488 (green fluorophore) Chicken anti-rabbit AF594 (red fluorophore) Chicken anti-rabbit AF546 (red fluorophore) Chicken anti-rabbit AF605 (cyan fluorophore) Chicken anti-goat HRP Chicken anti-goat AF488 (green fluorophore) Chicken anti-goat AF594 (red fluorophore) Chicken anti-goat AF546 (red fluorophore) Chicken anti-goat AF605 (cyan fluorophore)

Rabbit anti-guinea pig HRP Rabbit anti-guinea pig AF488 (green fluorophore) Rabbit anti-guinea pig AF594 (red fluorophore) Rabbit anti-guinea pig AF546 (red fluorophore) Rabbit anti-guinea pig AF605 (cyan fluorophore) Chicken anti-guinea pig HRP Chicken anti-guinea pig AF488 (green fluorophore) Chicken anti-guinea pig AF594 (red fluorophore) Chicken anti-guinea pig AF546 (red fluorophore) Chicken anti-guinea pig AF605 (cyan fluorophore)

Design one or more panels that allow you distinguish between blood and lymphatic vessels

and that allow you to determine whether alpha or beta cells are vascularized (the vessels are in contact with the cells)

22. Discuss these FACS plot. In specific, tell me what are the effects of IL-7 vs. IL-12 vs. IL-12 + IL-

17 addition to the medium compared to the medium on IFNγ expression on CD4+ cells and on CD8+ cells (4)

23. Discuss these FACS plot. In specific, tell me what are the effects of growing A549 cells in

sphere (A549 sphere) vs. control (A549) on expression of CD133 (as measured by PE- conjugated antibody) and on CD90 (as measured by FITC-conjugated antibody) (4)

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