ESTABLISHING OF ANIMAL MODEL TRANSGENIC MICE TO SUPPORT

ESTABLISHING OF ANIMAL MODEL (TRANSGENIC MICE) TO SUPPORT THE RESEARCH IN CANCER Ahmad Aulia Jusuf Department of Histology Faculty of Medicine University of Indonesia 2009 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009

Agenda ® Introduction ® Definition and useful of transgenic mice ® The method of producing transgenic mice ® The using of transgenic mice in studying the carcinogenesis and in searching the substance or chemical agent including the herb in the treatment of cancer Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 2

Introduction ® Transgenic mice v Used widely in the biomedical research v History 2, 3, 4, 5, 6 q Palmitter dan Brinster (1981) Ø Ø q q Generating the transgenic mice firstly Insertion of viral thymidine kinase gene into the fertilized eggs Palmitter (1986), Jaenisch (1988) dan Hanahan (1989) Gordon (1983), Hogan 1994 Injection of gene into pronuclear fertilized egg ___________________________________________________________ Ø _______________2 Jusuf, A. A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various human disease. Medical Journal Of Indonesia. 1998: 7; 2 : 55 -64 3 Brinster R, Chen H, Trumbauer M et al : Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 1981: 27: 223 -231 4 Palmitter R. D and Brinster R. L: Germ-line transformation of mice. Annu. Rev. Genet 1986: 20; 465 -499 5 Jaenisch R : Transgenic animals. Science 1988: 240; 14681474 6 Hanahan D. : Transgenic mice as probes into complex system. Science 1989: 246; 1265 -1275 7 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 3

Introduction ® Transgenic mice v Used as animal model to study 1, 2, 3, 10 The regulation of gene associated with the development of the tissue q The function of specific gene involved in the tissue development q Regulation of oncogenes and tumor suppressor q The effects of substances or chemical substance in therapy of cancer ___________________________________________________________ q _______________2 Jusuf, A. A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various human disease. Medical Journal Of Indonesia. 1998: 7; 2 : 55 -64 3 Brinster R, Chen H, Trumbauer M et al : Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 1981: 27: 223 -231 4 Palmitter R. D and Brinster R. L: Germ-line transformation of mice. Annu. Rev. Genet 1986: 20; 465 -499 5 Jaenisch R : Transgenic animals. Science 1988: 240; 14681474 6 Hanahan D. : Transgenic mice as probes into complex system. Science 1989: 246; 1265 -1275 7 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 4

Definition and useful of transgenic mice ® Transgenic Mice 1, 2 v An organism (mice) whose genome q q has been artificially modified by means of genetic engineering has to be transmitted hereditary expression of exogenous gene v over-expression of endogenous gene v _____________________________________________________________1 Aguzzi A, Brandner S, Isenmann S, Steinbach JP, and Sure U : Transgenic and gene disruption techniques in the study of neurocarcinogenesis. Glia 1995: 15: 348 -364 2 Jusuf, A. A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various human disease. Medical Journal of Indonesia. 1998: 7; 2 : 55 -64 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 5

Definition and useful of transgenic mice ® DNA fragments or gene inserted into the cell v v v ligated end to the genome by the intracellular ligation enzyme randomly integrated into a site in the chromosome arranged in the tandem manner 2, 9 gene ______________________________________________________________ 2 Jusuf, A. A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various human disease. Medical Journal of Indonesia. 1998: 7; 2 : 55 -64 9 Albert, B. , Bray, D. , Lewis, J. , Raff, M. , Roberts, K. , Watson, J. D. (1994), Cellular Mechaninsm of development in Molecular Biology of The Cell. , 3 rd Ed. , Garland Publishing, New York and London, pp. Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 6

Definition and useful of transgenic mice ® The useful of transgenic mice 2, 3, 10 v Animal Model q q to study the regulation of gene associated with the development of the tissue to study the function of specific gene involved in the tissue development and the phenotype of those genes in the tissue to study regulation of genes involved in the oncogenesis The effects of substances or chemical substance in therapy of cancer _____________________________________________________________ 2 Jusuf, A. A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various human disease. Medical Journal Of Indonesia. 1998: 7; 2 : 55 -64 3 Brinster R, Chen H, Trumbauer M et al : Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 1981: 27: 223 -231 10 Nagy A, Gertsenstein M, Vintersen K, Behringer R, Manipulating the mouse embryo: A laboratory manual. 3 rd Ed New York: Cold Spring Harbor: 2003 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 7

The procedures of producing transgenic mice ® The processes of making transgenic mice v difficult and complicated v take time, energy and high cost ® 2 methods 19, 20 v Pronuclear microinjection v Embryonic Stem (ES) cell electroporation and subsequent blastocyst injection Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 8

Method of making transgenic mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 9

The procedures of producing transgenic mice ® The steps v preparation of transgen which will be injected into pronuclear of fertilized eggs v preparation of mice v preparation of tools and material v preparation of detection system Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 10

The procedures of producing transgenic mice ® Preparation of transgen which will be injected into pronuclear of fertilized eggs v DNA fragment or transgene q q promoter, complete protein coding region, < satu intron dan polyadenylation site 11 the length of DNA transgene is unlimited Ø Ø from several kilo base pair (Kbp) until 1000 kb (Lamb et 1999)13 ideal length of DNA fragment is 2 - 20 Kb amplification of DNA fragment by using PCR q gene construction, amplification and purification the DNA fragment by using the molecular biology protocols q has to be purified and free from the sequence of prokaryotic vector 12 ___________________________________ q concentration of 1 -2 µg/ml Brinster R, Allen J, Behringer. R, Gelimas R et al. : Intron s increase transcriptional efficiency in transgenic mice. Proc Natl. q 11 Acad. Sci. USA 1988: 85; 836 -40 K, Magram J, Raphael K, Radice G, Lacy E and Constantini F. Spesific expression of a foreign beta globin gene in eryrthroid cells of transgenic mice. Nature. 1985: 314: 377 -380 13 Lamb B. T, Bardel K. A, Kulnane L. S. Anderson J. J, Holtz G, Wagner S. L, Sisodia S. S, and Hoeger E. J. Amyloid production and deposition in mutant amyloid precursor protein and presenilin-1 yeast artificial chromosome transgenic mice. Natl. Neurosci. 1999: 2; 695 -697 19 Transgenic mice, diunduh dari htpp: //cancer ucsd. edu/tgm/pronuclear. asp 12 Chada Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 11

The procedures of producing transgenic mice ® Preparation of transgen which will be injected into pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 12

The procedures of producing transgenic mice ® Preparation of transgen which will be injected into pronuclear of fertilized eggs v Endogenous transgene insertion of oligonucleotide in the untranslated region of DNA fragment 14, 15 q insertion the small gene that express small RNA in transgene 16 q fusion of protein transgen with protein Green Fluorescent Protein (GFP) encoded by gene reporter alkaline phosphatase 17 ___________________________________ q 14 Peschon J. J, Behringer R. R, Brinster R. L, and Palmiter R. D. Spermatide-specific expression of protamine 1 in transgenic mice. Proc Natl. Acad. Sci. USA. 1987: 84; 5316 -5319 15 Shi Y, Son H. J, Shahan K, Rodriguez M, Constantini F and Derman E. Silent genes in the mouse major urinary protein gene family. Proc Natl. Acad. Sci. USA. 1989: 86; 4584 -4588 16 Krumlauf R, Hammer R. E, Tilghman S. M, and Brinster R. L Developmental regulation of alpha-fetoprotein genes in transgenic mice. Mol. Cell. Biol. 1985: 5; 1639 -1648. 17 van Roessel P and Brand A. H. Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat. Cell. Biol. 2002: 4; 15 -20 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 13

The procedures of producing transgenic mice ® Preparation of transgen which will be injected into pronuclear of fertilized eggs v Efficiency of transgene integration 8, 10 q Shape of DNA Ø q Concentration of DNA Ø Ø Ø q >> in the higher concentartion Too high DNA concentration increase the toxicity for zygote Concentration of DNA : 1. 0 -2 nanogram/microliter Purity of DNA Ø q >> in the linearized DNA Transgene has to be free from contamination (remaining phenol, ethanol or enzymes) Buffer solution Ø TE buffer containing 1 m. M EDTA. ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 10 Nagy A, Gertsenstein M, Vintersen K, Behringer R, Manipulating the mouse embryo: A laboratory manual. 3 rd Ed New York: Cold Establishing of Animal Model Spring Harbor: 2003 Wednesday, Agust 12, 009 (transgenic mice)/AAJ/Histo. FKUI/2009 14

The procedures of producing transgenic mice ® Preparation of Mice 8 v Superovulated female mice q q q to get the fertilized eggs >> Follicular development is stimulated with Pregnant Mare’s Serum Gonadotrophin (PMSG) Ovulation is stimulated with Human Chorionic Gonadotrophin (HCG) Age : 4 -8 weeks Mate with fertile stud mice Check the copulation plug after mating ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 15

The procedures of producing transgenic mice ® Preparation of Mice 8 v Fertile stud male mice q q v Mate with superovulated female mice Age 6 -8 weeks with good reproduction performance Sterile male mice q q q has been vasectomized mate with pseudopregnant female mice Age of >> 8 weeks ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 16

Vasectomization in the Sterile Male Mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 17

The procedures of producing transgenic mice ® Preparation of Mice 8 v Pseudopregnant female mice age of 6 -8 weeks with big infundibulum q mate with sterile male mice q check the copulation plug q receives the fertilized eggs that have contained the transgenes q ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 18

The procedures of producing transgenic mice ® Preparation of tools and materials 8 v v Disposible syringe Pregnant Mare’s Serum Gonadotrophin (PMSG) hormone Human Chorionic Gonadotrophin (HCG) hormone Culture solution q q v v v D-PBS or other solutions that have added bovine serum albumin (BSA), piruvate acid antibiotic (penicillin and streptomycin) Culture solutions that contain hyaluronidase enzyme Incubator (37 C, humidity 95% and 5% CO 2 Surgical set Alcohol 70% stereomicroscope Mouth controlled pipette 35 mm petri dish ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 19

The procedures of producing transgenic mice ® Preparation of tools and materials 8 v Holding pipette q q v Borosilicate glass capilary mechanical pipette puller Injection pipette q q Glass capillary tubing dengan internal glass filament mechanical pipette puller ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 20

The procedures of producing transgenic mice ® Preparation of tools and materials 8 v Microscope with micromanipulator q q q v Paraffin oil Petri dish injection chamber Transgen Detection of transgene q q q Southern blot Northern blot Western Blot ___________________________________ 8 Hogan B, Constantini. F, Lacy E. : Manipulating the mouse embryos: A laboratory manual. 2 nd Ed. New York : Cold Spring Harbor: 1994 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 21

Steps in Production of transgenic mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 22

Steps in Production of transgenic mice ® Injection of hormone PMS and h. HCG Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 23

Steps in Production of transgenic mice ® Isolation and collecting of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 24

Steps in Production of transgenic mice ® Isolation and collecting of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 25

Steps in Production of transgenic mice ® Isolation and collecting of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 26

Steps in Production of transgenic mice ® Isolation and collecting of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 27

Steps in Production of transgenic mice ® Isolation and collecting of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 28

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 29

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 30

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 31

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 32

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 33

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 34

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 35

Steps in Production of transgenic mice ® Injection of transgene into male pronuclear of fertilized eggs Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 36

Steps in Production of transgenic mice ® Transfer of fertilized eggs containing transgene into oviduct of pseudopregnant female mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 37

Steps in Production of transgenic mice ® Transfer of fertilized eggs containing transgene into oviduct of pseudopregnant female mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 38

Steps in Production of transgenic mice ® Transfer of fertilized eggs containing transgene into oviduct of pseudopregnant female mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 39

Steps in Production of transgenic mice ® Maintain the “candidate” for transgenic mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 40

Steps in Production of transgenic mice ® Detection of Transgene in the candidate of transgenic mice Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 41

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice Patogenesis and patophysiology of cancer v Regulation of genes belongs to oncogene and tumor suppressor v Abnormality of tissue v Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 42

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice v Green et. al 21 q q q Transgenic mice as a model to study carcinoma of prostat SV- 40 large tumor antigen coding region with prostate-specific rat probasin as its promoter Phenotype of transgenic mice Ø Ø Ø ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia expression of gene p 53 >> expression of receptor androgen in the prostat << ___________________________________ 21 Greenberg NM, Demayo FJ, Finegold M, et al Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA. 1995: 92: 3439 -43. Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 43

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice v Janz et. al 22 q q v transgenic mice for Burkitt’s lymphoma MYC transgene under its promoter Rao et. al. 23 q transgenic mice to study the breast cancer Ø transgene of oncogen c-neu ___________________________________ 22 Janz, HC Morse III, et al. Burkitt's lymphoma in the mouse. Journal of Experimental Medicine. 2000: 92: 8: 1183 -90 23 Rao G. N. , Ney, E. , Herbert R. A. Influence of diet on mammary cancer in transgenic mice bearing an oncogene expressed in mammary tissue. Breast Cancer Research and treatment. 1997: 45: 2: 149 -158(10) Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 44

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice v Kim et. al. 24 q insertion entire HBx gene under its own regulatory elements Ø q to study the role of that gene in patogenesis of hepatoma results Ø progressive histopathological changes specifically in the liver, beginning with multifocal areas of altered hepatocytes, followed by the appearance of benign adenomas, and proceeding to the development of malignant carcinomas ___________________________________ 24 Kim C. M. , Koike. K. , Saito, I. , Miyamura, T. , Jay, G. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature. 1991: 351: 317 - 320 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 45

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice that express the protein oncogen E 6 and E 7 of Human Papilloma Virus (HPV) v Arbeit et. al 25 q Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites including ear and truncal skin, face, snout and eyelids, and anus q The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance. ___________________________________ 25 Arbeit, J. M. , Munger, K. , Howley, P. M. , Hanahan, D. Progessive squamous epithelial neoplasia in K 14 -human papillomavirus type 16 transgenic mice. J Virol. 1994: 68(7): 4358 -4368 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 46

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice that express the protein oncogen E 6 and E 7 of Human Papilloma Virus (HPV) v Riley et. al. 26 q Phenotype mimic to cervical carcinoma ___________________________________ 26 Riley, R. R. , Duensing, S. , Brake, T. , Münger, K. , Lambert, P. F. , Arbeit, J. M. Dissection of Human Papillomavirus E 6 and E 7 Function in Transgenic Mouse Models of Cervical Carcinogenesis. Cancer Research. 2003: 63: 48624871 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 47

Fig. 2. Histopathology of the CIN scoring system based on the K 14 -HPV mouse. The murine CIN grading system depicting degree of reproductive tract squamous epithelial dysplasia and neoplastic progression in estrogen treated K 14 -HPV transgenic mice (panels 2– 6) and nontransgenic controls (panel 1). The squamous epithelium in nontransgenic mice is hyperplastic and mitotic figures are present only in basal cells (black arrow, panel 1). CIN I lesions (panel 2) evidence occasional anaplastic squamous epithelial cells that are persistent in the upper differentiated layers (white arrowhead). CIN II lesions (panel 3) display epithelial cell projections into the stroma (papillation, black arrows), and cells with an additional degree of nuclear anaplasia (white arrowheads). CIN III lesions (panel 4) are similar to CIN II except the frequency of anaplastic squamous epithelial cells is increased. CIS lesions (panel 5) are distinguished by additional cytological squamous epithelial cell progression and more irregularity of the epithelialstromal border. Squamous cell carcinoma (SCC; panel 6) retain a well to moderate degree of squamous epithelial differentiation and invade into the stroma without evidence of encirclement by basement membrane (black arrows). SCCs often arise from glandular squamous metaplasia at the transformation ___________________________________ 26 Riley, R. R. , Duensing, S. , Brake, T. , Münger, K. , Lambert, P. F. , Arbeit, J. M. Dissection of Human Papillomavirus E 6 zone (black arrowhead). Bar, panels 1– 5 = 20 µm; panel 6 = 100 µm. and E 7 Function in Transgenic Mouse Models of Cervical Carcinogenesis. Cancer Research. 2003: 63: 48624871 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 48

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice that express the protein oncogen E 6 and E 7 of Human Papilloma Virus (HPV) v Searle et. al. 27 solitary gland within the gastric mucosa became colonized with small dysplastic cells q the abnormal cell population spread within the glandular mucosa, invaded the submucosa and outer muscular wall of the stomach, and commonly metastasized to local ___________________________________ lymph nodes and the liver Searle, P. F. , Thomas, D. P. , Faulkner, K. B. , Tinsley, J. M. Stomach Cancer in Transgenic Mice Expressing Human q 27 Papillomavirus Type 16 Early Region Genes From a Keratin Promoter. J Gen Virol. 1994: 75: 1125 -1137 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 49

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice v Venkateswaran et. al 28 q Using the lady transgenic mice Ø antioxidants (vitamin E, selenium, and lycopene) in the diet dramatically inhibits prostate cancer development and increases the disease free survival ___________________________________ 28 Venkateswaran, V. , Fleshner, N. E. , Sugar, L. M. , Klozt, L. H. Antioxidants block prostate cancer in lady transgenic mice. Cancer Research. 2004: 64: 5891 -96. Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 50

The using of transgenic mice in studying the carcinogenesis ® Transgenic mice v Kato et. al 29 q metallothionein-I (MT)/ret transgenic mouse and melanoma cell (Mel-ret) transgenic mice Ø herbal medicine sho-saiko-to Inhibits Growth and Metastasis of Malignant Melanoma Primarily Developed in ret-Transgenic Mice ___________________________________ 29 Kato, M. , Liu, W. , Yi, H. , Asai, N. , Hayakawa, A. , Kozaki, K. , Takahashi, M. , Nakashima, I. The Herbal Medicine Sho- saiko-to Inhibits Growth and Metastasis of Malignant Melanoma Primarily Developed in ret-Transgenic Mice. Journal of Investigative Dermatology. 1998: 111: 640– 644 Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 51

Summary ® ® ® Transgenic mice is an organism (mice) whose genome has been artificially modified by means of genetic engineering has to be transmitted hereditary The processes of making transgenic mice are difficult, complicated, take time and energy and high cost Transgenic mice functions as an animal Model q q to study the regulatin of gene associated with the development of the tissue to study the function of specific gene involved in the tissue development and the phenotype of those genes in the tissue to study regulation of genes involved in the oncogenesis The effects of substances or chemical substance in therapy of cancer Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 52

Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 53

Wednesday, Agust 12, 009 Establishing of Animal Model (transgenic mice)/AAJ/Histo. FKUI/2009 54