establishing of animal model (transgenic mice) to support the research in cancer

ESTABLISHING OF ANIMAL MODEL (TRANSGENIC MICE) TO SUPPORT THE

RESEARCH IN CANCERAhmad 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

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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



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Introduction Transgenic mice

Used widely in the biomedical research History2,3,4,5,6

Palmitter dan Brinster (1981) 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

__________________________________________________________________________________________________________________________________________________ 2Jusuf, 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 3Brinster 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: 223-2314Palmitter R.D and Brinster R.L: Germ-line transformation of mice. Annu. Rev. Genet 1986: 20; 465-4995Jaenisch R : Transgenic animals. Science 1988: 240; 146814746Hanahan D. : Transgenic mice as probes into complex system. Science 1989: 246; 1265-12757Hogan B, ConstantiniF, Lacy E. : Manipulating the mouse embryos: A laboratory manual.2nd Ed .New York : Cold Spring Harbor: 1994



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Introduction Transgenic mice

Used as animal model to study 1,2,3,10

The regulation of gene associated with the development of the tissue

The function of specific gene involved in the tissue development

Regulation of oncogenes and tumor suppressor The effects of substances or chemical substance

in the therapy of cancer

__________________________________________________________________________________________________________________________________________________ 2Jusuf, 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 3Brinster 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: 223-2314Palmitter R.D and Brinster R.L: Germ-line transformation of mice. Annu. Rev. Genet 1986: 20; 465-4995Jaenisch R : Transgenic animals. Science 1988: 240; 146814746Hanahan D. : Transgenic mice as probes into complex system. Science 1989: 246; 1265-12757Hogan B, ConstantiniF, Lacy E. : Manipulating the mouse embryos: A laboratory manual.2nd Ed .New York : Cold Spring Harbor: 1994



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Definition and useful of transgenic mice Transgenic Mice1,2

An organism (mice) whose genome has been artificially modified by means of

genetic engineering has to be transmitted hereditary

expression of exogenous gene over-expression of endogenous gene

______________________________________________________________________________________________________________________________________________________ 1Aguzzi A, Brandner S, Isenmann S, Steinbach JP, and Sure U : Transgenic and gene disruption techniques in the study of neurocarcinogenesis. Glia 1995: 15: 348-3642Jusuf, 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



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Definition and useful of transgenic mice

DNA fragments or gene inserted into the cell ligated end to end to the genome by the intracellular ligation enzyme randomly integrated into a site in the chromosome arranged in the tandem manner 2,9

________________________________________________________________________________________________________________________________________________________ 2Jusuf, 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 9Albert, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D. (1994), Cellular Mechaninsm of development in Molecular Biology of The Cell., 3rd Ed., Garland Publishing, New York and London, pp.

gene gene gene gene



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Definition and useful of transgenic mice

The useful of transgenic mice2,3,10

Animal Model 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 the therapy of cancer

_____________________________________________________________________________________________________________________________________________________ 2Jusuf, 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 3Brinster 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: 223-23110Nagy A, Gertsenstein M, Vintersen K, Behringer R, Manipulating the mouse embryo: A laboratory manual. 3rd Ed New York: Cold Spring Harbor: 2003



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The procedures of producing transgenic mice

The processes of making transgenic mice difficult and complicated take time, energy and high cost

2 methods19,20

Pronuclear microinjection Embryonic Stem (ES) cell electroporation and

subsequent blastocyst injection



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Method of making transgenic mice



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The steps preparation of transgen which will be injected into

pronuclear of fertilized eggs preparation of mice preparation of tools and material preparation of detection system



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The procedures of producing transgenic mice Preparation of transgen which will be injected into pronuclear

of fertilized eggs DNA fragment or transgene

promoter, complete protein coding region, < satu intron dan polyadenylation site11 the length of DNA transgene is unlimited

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 gene construction, amplification and purification the DNA fragment by using the

molecular biology protocols has to be purified and free from the sequence of prokaryotic vector12

concentration of 1-2 µg/ml

______________________________________________________________________11 Brinster R, Allen J, BehringerR, Gelimas R et al.: Intron s increase transcriptional efficiency in transgenic mice. Proc Natl. Acad.Sci.USA 1988: 85; 836-4012Chada 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: 377-38013Lamb 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; 695-69719Transgenic mice, diunduh dari htpp://cancer ucsd.edu/tgm/pronuclear.asp



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The procedures of producing transgenic mice Preparation of transgen which will be injected into pronuclear of

fertilized eggs



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Preparation of transgen which will be injected into pronuclear of fertilized eggs Endogenous transgene

insertion of oligonucleotide in the untranslated region of DNA fragment14,15

insertion the small gene that express small RNA in transgene16

fusion of protein transgen with protein Green Fluorescent Protein (GFP) encoded by gene reporter alkaline phosphatase17

______________________________________________________________________14Peschon 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; 5316-531915Shi 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; 4584-458816Krumlauf 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; 1639-1648.17van 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



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The procedures of producing transgenic mice Preparation of transgen which will be injected into pronuclear of

fertilized eggs Efficiency of transgene integration8,10

Shape of DNA >> in the linearized DNA

Concentration of DNA >> in the higher concentartion Too high DNA concentration increase the toxicity for zygote Concentration of DNA : 1.0-2 nanogram/microliter

Purity of DNA Transgene has to be free from contamination (remaining phenol, ethanol or enzymes)

Buffer solution TE buffer containing 1mM EDTA.

______________________________________________________________________8Hogan B, ConstantiniF, Lacy E. : Manipulating the mouse embryos: A laboratory manual.2nd Ed .New York : Cold Spring Harbor: 199410Nagy A, Gertsenstein M, Vintersen K, Behringer R, Manipulating the mouse embryo: A laboratory manual. 3rd Ed New York: Cold Spring Harbor: 2003



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Preparation of Mice8

Superovulated female mice 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

______________________________________________________________________8Hogan B, ConstantiniF, Lacy E. : Manipulating the mouse embryos: A laboratory manual.2nd Ed .New York : Cold Spring Harbor: 1994



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Fertile stud male mice Mate with superovulated female mice Age 6-8 weeks with good reproduction performance

Sterile male mice has been vasectomized mate with pseudopregnant female mice Age of >> 8 weeks




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Vasectomization in the Sterile Male Mice



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Pseudopregnant female mice age of 6-8 weeks with big infundibulum mate with sterile male mice check the copulation plug receives the fertilized eggs that have contained the

transgenes




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The procedures of producing transgenic mice Preparation of tools and materials8

Disposible syringe Pregnant Mare’s Serum Gonadotrophin (PMSG) hormone Human Chorionic Gonadotrophin (HCG) hormone Culture solution

D-PBS or other solutions that have added bovine serum albumin (BSA), piruvate acid and antibiotic (penicillin and streptomycin)

Culture solutions that contain hyaluronidase enzyme Incubator (37C, humidity 95% and 5% CO2 Surgical set Alcohol 70% stereomicroscope Mouth controlled pipette 35 mm petri dish




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Preparation of tools and materials8

Holding pipette Borosilicate glass capilary mechanical pipette puller

Injection pipette Glass capillary tubing dengan

internal glass filament mechanical pipette puller




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Preparation of tools and materials8

Microscope with micromanipulator Paraffin oil Petri dish injection chamber Transgen

Detection of transgene Southern blot Northern blot Western Blot




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Steps in Production of transgenic mice



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Injection of hormone PMS and hHCG



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Steps in Production of transgenic mice Isolation and collecting of fertilized eggs



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Steps in Production of transgenic mice Injection of transgene into male pronuclear of fertilized eggs



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Injection of transgene into male pronuclear of fertilized eggs



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Steps in Production of transgenic mice Injection of transgene

into male pronuclear of fertilized eggs



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Steps in Production of transgenic mice Transfer of fertilized eggs containing transgene into oviduct of

pseudopregnant female mice



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Maintain the “candidate” for transgenic mice



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Steps in Production of transgenic mice Detection of Transgene in the candidate of transgenic mice



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The using of transgenic mice in studying the carcinogenesis

Transgenic mice Patogenesis and patophysiology of cancer Regulation of genes belongs to oncogene and tumor

suppressor Abnormality of tissue



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Transgenic mice Green et.al21

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

ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia expression of gene p53 >> expression of receptor androgen in the prostat <<

_____________________________________________________________________21Greenberg NM, Demayo FJ, Finegold M, et al Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA. 1995:92: 3439-43.



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Transgenic mice Janz et.al22

transgenic mice for Burkitt’s lymphoma MYC transgene under its promoter

Rao et.al.23 transgenic mice to study study the breast cancer

transgene of oncogen c-neu

______________________________________________________________________22Janz, HC Morse III, et al. Burkitt's lymphoma in the mouse. Journal of Experimental Medicine.2000: 92:8:1183-90 23Rao 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)



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The using of transgenic mice in studying the carcinogenesis Transgenic mice

Kim et.al.24

insertion entire HBx gene under its own regulatory elements to study the role of that gene in patogenesis of hepatoma

results 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

______________________________________________________________________24Kim 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



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Transgenic mice that express the protein oncogen E6 and E7 of Human Papilloma Virus (HPV) Arbeit et.al25

Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites including ear and truncal skin, face, snout and eyelids, and anus

The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance.

_____________________________________________________________________25Arbeit, J.M., Munger,K., Howley,P.M.,Hanahan,D. Progessive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice. J Virol. 1994: 68(7): 4358-4368



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Transgenic mice that express the protein oncogen E6 and E7 of Human Papilloma Virus (HPV) Riley et.al.26

Phenotype mimic to cervical carcinoma

_____________________________________________________________________26Riley, R.R., Duensing,S., Brake,T., Münger,K., Lambert, P.F., Arbeit, J.M. Dissection of Human Papillomavirus E6 and E7 Function in

Transgenic Mouse Models of Cervical Carcinogenesis. Cancer Research.2003: 63:4862-4871



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Fig. 2. Histopathology of the CIN scoring system based on the K14-HPV mouse. The murine CIN grading system depicting degree of reproductive tract squamous epithelial dysplasia and neoplastic progression in estrogen treated K14-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 epithelial-stromal 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 zone (black arrowhead). Bar, panels 1–5 = 20 µm; panel 6 = 100 µm.

_____________________________________________________________________26Riley, R.R., Duensing,S., Brake,T., Münger,K., Lambert, P.F., Arbeit, J.M. Dissection of Human Papillomavirus E6 and E7 Function in

Transgenic Mouse Models of Cervical Carcinogenesis. Cancer Research.2003: 63:4862-4871



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Transgenic mice that express the protein oncogen E6 and E7 of Human Papilloma Virus (HPV) Searle et.al.27

solitary gland within the gastric mucosa became colonized with small dysplastic cells

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

_____________________________________________________________________27Searle, P.F., Thomas, D.P., Faulkner, K.B., Tinsley, J.M. Stomach Cancer in Transgenic Mice Expressing Human Papillomavirus Type 16

Early Region Genes From a Keratin Promoter. J Gen Virol. 1994: 75: 1125-1137



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Transgenic mice Venkateswaran et.al28

Using the lady transgenic mice antioxidants (vitamin E, selenium, and lycopene) in the diet dramatically

inhibits prostate cancer development and increases the disease free survival

_____________________________________________________________________28Venkateswaran, V., Fleshner, N.E., Sugar,L.M., Klozt, L.H. Antioxidants block prostate cancer in lady transgenic mice. Cancer

Research .2004:64:5891-96.



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Transgenic mice Kato et.al29

metallothionein-I (MT)/ret transgenic mouse and melanoma cell (Mel-ret) transgenic mice

herbal medicine sho-saiko-to Inhibits Growth and Metastasis of Malignant Melanoma Primarily Developed in ret-Transgenic Mice

_____________________________________________________________________29Kato,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



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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 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 the therapy of cancer



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establishing of animal model (transgenic mice) to support the research in cancer

Documents

transgenic animals

brinster r

organism mice

injection of gene

jaenisch r

palmitter r

fusion gene

regulation of gene