Generating Mouse Models of Pancreatic CancerGenerating Mouse Models of Pancreatic Cancer

Aom Isbell

http://www2.massgeneral.org/cancerresourceroom/types/gi/index.asp

Spring/Summer 1 , 2012

Alexandros Tzatsos, MD PhDAlexandros Tzatsos, MD PhD

Bardeesy Lab: Goals and Objectives Bardeesy Lab: Goals and Objectives Objective: Understanding the genetic program for Pancreatic Ductal Adenocarcinoma (PDAC) initiation and progress -*Approx. 44K new cases in US in 2012 -*4.6% survival rate average (beyond 5 years) Goal: Engineering conditional knockout and overexpression ngineering conditional knockout and overexpression mouse models to study initiation and maintenance of PDAC mouse models to study initiation and maintenance of PDAC

Aim: To generate mouse models that mimic the human disease in order to be used in therapeutic drug screening

www.bing.com/images/search?q=knockdown+mouse&view

*National Cancer Institute website statistics

The Pancreas has an exocrine and endocrine function●Endocrine- hormones (insulin, glucagon) secreted directly into the bloodstream, accounts for about 10% pancreatic cancers

●Exocrine- digestive enzymes produced by epitheleal duct cells secreted through ducts into digestive tract, accounts for 90% of pancreatic cancers and, unlike endcorine, is extremely aggressive

http://www.gopetsamerica.com/anatomy/illustrations/pancreas.jpg

Endocrine 10% Exocrine 90%

Pancreatic Cancers

PanINs, pancreatic interepithelial neoplastic lesions

PDAC, pancreatic ductal adenocarcinoma

In 90% of PDACs mutated KRAS oncogenesare present

KRAS is normally inactive if bound to GDP(guanosine diphosphate) but mutated KRAS remains constitutively active which leads tocell proliferation and migration and DNA synthesis

Constitutively activated KRAS drives the progression of PDAC

Lysine Specific Demethylase 2B: enzyme encoded by the KDM2B Gene

●Regulates epithelial cell differentiation during development●Overexpression promotes bypass of senescence and immortalizes cells

●Upregulation is associated with advanced disease in PDAC

KDM2B cooperates with KRAS to drive PDAC

●Deregulates cell proliferation downstream of KRAS●Blocks tumor suppressors (such as p16) allowing KRAS to cause proliferated cell mutation

*A. Tzatsos, P. Paskaleva et al.

*

Project TitleProject Title Genotyping of GEMM Genotyping of GEMM (genetically engineered mouse models)(genetically engineered mouse models)

Using Polymerase Chain Reaction to test for the presence of Using Polymerase Chain Reaction to test for the presence of Cre, LoxP, and KRAS in selected mouse models Cre, LoxP, and KRAS in selected mouse models --Cre recombinaseCre recombinase: enzyme that carries out site specific DNA: enzyme that carries out site specific DNA recombination (recognizes LoxP sequence)recombination (recognizes LoxP sequence) --LoxPLoxP: target sequence of DNA (locus) : target sequence of DNA (locus)

Using Gel Electrophoresis to visualize results Using Gel Electrophoresis to visualize results Mice are being engineered to conditionally overexpress or ablate Mice are being engineered to conditionally overexpress or ablate KDM2B (Dox inducibility)KDM2B (Dox inducibility)

Mice that possess these genes can be bred to create Mice that possess these genes can be bred to create Homozygous (knockout or overexpression) models and Homozygous (knockout or overexpression) models and examined through histological methods to further determine examined through histological methods to further determine the roles these genes have in disease initiation and the roles these genes have in disease initiation and progressionprogression

http://en.wikipedia.org/wiki/Cre-Lox_recombination

AblationAblation

Proof of PrincipleProof of Principle KRAS X KDM2B (Deletion)

Al1 + + -Al2 + - -

PDX1 Cre X

LoxP KDM2B LoxP

PDX1 is a tissue specific promoter (Pancreas)provides Spatial Control over transgene expression. -Cre will only be active in pancreas cellsGATA1 is whole body promoter, will promote expression in all cells types. Successful cross breeding will yield a mouse Successful cross breeding will yield a mouse with both alleles of the KDM2B gene deleted with both alleles of the KDM2B gene deleted Homozygous Knockout Homozygous Knockout

HomozygousHomozygous Knockout (-/-)Knockout (-/-) yields little PDAC development

HeterozygousHeterozygous Wild Type/Knockout (+/-)Wild Type/Knockout (+/-) yields Intermediate development of PDACHomozygousHomozygous Wild Type (+/+)Wild Type (+/+) yields extensive PDAC development

GATA1 Cre X Or

Techniques in Molecular Biology, Spring '11 Lecture Slide

The Targeting Vector (DNA Construct)

1. Homologous DNA that matches the gene (orangeorange)2. Positive selection marker-neor (neomycin resistance) ensures the vector is inserted (redred)

Techniques in Molecular Biology, Spring '11 Lecture slides

2.

Techniques in Molecular Biology, spring '11 lecture slides

HypothesisHypothesis: : Overexpression of KDM2B will enhance Overexpression of KDM2B will enhance KRAS' ability to form PDAC, whereas KDM2B genetic KRAS' ability to form PDAC, whereas KDM2B genetic deletion will inhibit PDAC formation in the context of KRASdeletion will inhibit PDAC formation in the context of KRAS

Materials and MethodsMaterials and Methods

●Extract genomic DNA -Alkaline Lysis Buffer (NaOH, dH2O,0.5M EDTA pH 8.0,- -ethylenediaminetetraacetic acid) -Vortex -Neutralization Buffer (40mM Tris-HCl) -PCR program

●PCR Master Mixes -10x Buffer -dH20 -MgCl2+ -Genomic DNA samples -dNTPs -Taq Polymerase -Specific Primers (Gabra/Kozak etc.)

Materials and Methods (continued)Materials and Methods (continued)

●Run PCR Program -Denaturing -Annealing -Extension -Exponential replication -Add dye and centrifuge

●Gel Preparation -Agarose powder -TBE (Tris base, boric acid, EDTA) -EtBr (ethidium bromide) -Gel castor and well comb

●Run Electrophoresis -Molecular ladder -Electrophoresis tub -TBE -Power Supply and cables

●Gel Imaging●Interpret results oceanexplorer.noaa.gov/explorations/04e

PCR gel image from 3/20/12 , testing for presence of LoxP in mice PCR gel image from 3/20/12 , testing for presence of LoxP in mice 259 to 274 259 to 274

+/+ +/--/-

259: (+/+) 260: (+/-) 261: (-/-)262: (-/-) 263: (+/-) 264: (-/-)265: (+/-) 266: (+/+) 267: (+/-)268: (+/+) 269: (-/-) 270: (+/-)271: (+/-) 272: (-/-) 273: (-/-)274: (+/-)

Wild type Heterozygous Homozygous deletion

LoxPLoxP==KDM2BKDM2B==LoxPLoxP

500 bp

100 bp

Gel ImageGel Image

The altered gene is The altered gene is bigger than(more base bigger than(more base pairs) the wild type due to pairs) the wild type due to the addition of the LoxP the addition of the LoxP and thus won't travel as and thus won't travel as far through the agarose far through the agarose gel matrixgel matrix

Molecular Ladder

Wild type at 200 BP

Altered gene at 300 BP

Control Lane (far right)

Results and ConclusionsResults and Conclusions●Gel image showed Gel image showed many of the mice tested carry the transgenemany of the mice tested carry the transgene, , thus can be used for further analysisthus can be used for further analysis

●One step in a larger procedureOne step in a larger procedure: further genotyping, breeding : further genotyping, breeding and histological examination of mouse models needed to and histological examination of mouse models needed to confirm hypothesisconfirm hypothesis

●Data shows mice # 261, 262, 264, 269, 272, and 273 all have the LoxP on both alleles of the target gene (homozygous deletionhomozygous deletion) making them suitable candidates forfurther cross-breeding and histological examination

●As MGH Cancer Center continues to examine and define the biology of PDAC through these methods, steps toward improved improved drug screening techniques and patient prognosisdrug screening techniques and patient prognosis will be made

●Personal data helped identify suitable candidatesdata helped identify suitable candidates for further transgene breeding and histological study

AcknowledgementsAcknowledgements

Alexandros Tzatsos, MD, PhD- team leader, mentor

Polina Paskaleva- mentor

Special Thanks to the Lab Personnel at Bardeesy Lab, MGH Cancer Center

Nabeel Bardeesy, PhD- principal investigator, Bardeeesy Lab

ReferencesReferences-“KDM2B controls tumorigenesis in pancreatic cancer via Polycomb-dependent and interdependent transcriptional programs”; A. Tzatsos, P. Paskaleva, F. Ferrari et al.-”Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling”; Z.Zhu, T. Zheng, C. Lee et al. ; Cell and Developmental Biology, vol 13, 2002-Techniques in Molecular Biology spring 2011, (course) Transfection, Transgenic Knockout, lecture slides Arthur Lambert-Biology of Cancer, fall 2011, (course), RAS cell transduction pathways, lecture slides, Arthur Lambert -Nature.com, Natural Protocols, v6n6 2011-National Cancer Institute.com, Pancreatic Cancer Big Picture-WHO.int , world health organization Pancreatic Cancer global statistics, pancreatic duct adenocarcinoma resources-Biology of Cancer, (textbook), Robert A. Weinberg, -Cancer.com, American Cancer Society, 'research and explore' resources

Image Sources

-Http://www2massgeneral.org/cancerresources/types/search?/gi/index.asp-Www.bing.com/imagessearch?9=knockdown+mouse&view-http://www.gopetsamerica.com/anatomy/illustrations/pancreas.jpg-J Gastroentero Hepatol © 2008 Buckwell Publishing-http://www.cancer.gov/cancertopics/types/pancreatic-Essential Cell Biology 3/e(©Garland Science 2010)-http://en.wikipedia.org/wiki/Cre-Lox recombination-http://bioscience.org/2006/v11/af/1799/fulltext.asp?bframe=figures.htm&doi=yes-www.nature.com/mt/journal/v9/n4-Techniques In Molecular Biology, Spring 2011: Transfection and Transgenic Knockdown Animals Lecture, Arthur Lambert-oceanexplorer.noaa.gov/explorations/04e

Doxycycline-inducible Models Doxycycline-inducible Models

Tet O Mini CMV Cre Tet O Mini CMV Crewww.nature.com/mt/journal/v9/n4

Doxycycline: a derivative of Tetracycline, induces a conformational change Tetracycline: an antibiotic produced by bacteriaTet O: tetracycline specific protein (operator), is a synthesis inhibitor when not bound by TATA: (transcriptional activator) binding protein that controls transcriptional flow Mini CMV: (promoter construct) derived from the human cytomegalovirus

Inducible models provide temporal control over transgene expression; some transgenes can be toxic to host organisms during gestation/embryogenesis

Gene Activation Using Cre-LoxPGene Activation Using Cre-LoxP

Cre recombinaseLoxP