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Characterization of ET1 ep-/- mouse line By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical Sciences Sep 25, 2009 By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical.

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Presentation on theme: "Characterization of ET1 ep-/- mouse line By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical Sciences Sep 25, 2009 By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical."— Presentation transcript:

1 Characterization of ET1 ep-/- mouse line By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical Sciences Sep 25, 2009 By Steven Ma Mentor: Dr. Arup Indra Pharmaceutical Sciences Sep 25, 2009

2 Why ET1 (endothelin-1)? Known growth factor – hyperproliferative pathologies: psoriasis and cancer? Endothelin “axis” has been shown to promote cancer progression (Bagnato et al. 2008) o Melanoma o Breast, prostate, brain, lung, bladder etc. Malignant melanoma is the leading cause of death (75%) in skin cancers - no curative measures yet (Jerant et al. 2000)

3 Endothelins What we know: 21 amino acid peptides Three isoforms, ET-1, ET-2, ET-3 Produced by keratinocytes at a basal level helin1_03.png What we don’t know: Autocrine functions in keratinocytes

4 Overview of Skin Many Layers 95% are keratinocytes Melanocytes o Secrete melanin – responsible for pigmentation (and tanning) o Derived from neural crest cells (same as neurons) Diferentiation Proliferation

5 Hypothesis ET1 has an autocrine effect on epidermal keratinocyte homeostasis, altering cell proliferation and differentiation. ET1 has a paracrine effect on melanocytes.

6 Getting ET-1 out of epidermis Bacteriophage P1 site-specific Cre-lox recombination Flank target genes with loxP sites Cre – Cyclic Recombinase Human – mice homology x

7 Techniques PCR: to confirm ET-1 ablation in epidermis Paraffin sections of skin treated with 4% PFA (paraformaldehyde) used for: Hematoxylin and eosin staining Immunohistochemistry Fontana Masson staining

8 Hematoxylin & eosin staining (HE) Stains nucleus blue and cytoplasm pink Popular method in histology, widely used in medical diagnosis, e.g. tumor biopsies Used to measure epidermal thickness

9 HF D E{E{ D E{E{ Data: epidermal thickness ET1 L2/L2 (wildtype) ET1 ep-/- (knockout) ET1 L2/L2 (wildtype)ET1 ep-/- Epidermal thickness (µm) No significance

10 Immunohistochemistry (IHC - with paraffin sections) Use antibodies to detect specific proteins in skin sections Purpose: analyze cell differentiation and proliferation Used Cy3 2ndary antibody

11 Epidermal Proteins Loricrin Keratin 10 (K10) Keratin 14 (K14) Late differentiating cells Early proliferating cells Ki 67Proliferation marker; present in all active cell phases (but not G 0 )

12 Red – Cy3 Blue – Dapi (all cells) ET1 L2/L2 (Wildtype)ET1 ep-/- Immunohistochemistry – differentiation D E K14 E D K10 D E Loricrin

13 Red – Cy3 Blue – Dapi (all cells) ET1 L2/L2 (Wildtype) ET1 ep-/- Immunohistochemistry – proliferation E D Ki 67 Counts (per 100 cells): ET1 L2/L2 (Wildtype) ET1 ep-/- (knockout)

14 Fontana-Masson Staining (FM) Stains all nuclei red, but melanocytes black “Melanin stain” Used to analyze paracrine effect of keratinocyte ET-1 on neighboring melanocytes

15 Fontana-Masson Stain ET1 L2/L2 (Wildtype)ET1 ep-/-

16 Fontana-Masson Stain: melanocyte count compared

17 Results thus far No significant autocrine effect on keratinocytes: – Proliferation – differentiation Significant effect on melanocyte proliferation Adult mice

18 Ongoing experiments UV treatment to new born mice – Skin samples at 0h, 24h, 48h time points – Fontana-Masson staining analysis qPCR for melanogensis factors in UV treated mice

19 Fontana-Masson Stain: UV treatment – 0 hour time point

20 Special thanks to: Dr. Kevin Ahern Dr. Arup Indra Steven Hyter Dr. Gitali Indra Xiaobo Liang Gaurav Bajaj Dan Coleman Zhixing Wang

21 References Jerant, A; Johnson, J; Sheridan, C; Cafferey, T. “Early Detection and Treatment of Skin Cancer.” American Family Physician. July 15, 2000 Bagnato, A; Spinella, F; Rosano, L. “The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can J Physiol Pharmacol. August, p473-84

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