Steps in Production of transgenic mice

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Dec 12, 2012 (4 years and 6 months ago)

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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

2

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

3

Introduction


Transgenic mice


Used widely in the biomedical research


History
2,3,4,5,6


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

_____________________________________________________________________________________________________________________________
___
______________
____
2
Jusuf, A.A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various

hu
man 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

ge
ne 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, ConstantiniF, Lacy E. : Manipulating the mouse embryos: A laboratory manual.2nd Ed .New York : Cold Spring Harbor: 1
994


Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

4

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

_____________________________________________________________________________________________________________________________
___
______________
____
2
Jusuf, A.A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of various

hu
man 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

ge
ne 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, ConstantiniF, Lacy E. : Manipulating the mouse embryos: A laboratory manual.2nd Ed .New York : Cold Spring Harbor: 1
994


Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

5

Definition and useful of transgenic mice


Transgenic Mice
1,2


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


_____________________________________________________________________________________________________________________________
___
__________________
____
1
Aguzzi A, Brandner S, Isenmann S, Steinbach JP, and Sure U : Transgenic and gene disruption techniques in the study of neuroc
arc
inogenesis. 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 variou
s h
uman disease. Medical
Journal of Indonesia. 1998: 7; 2 : 55
-
64


Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

6

Definition and useful of transgenic mice


DNA fragments or gene inserted into the cell


l
igated 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

_____________________________________________________________________________________________________________________________
___
_____________________
___

2
Jusuf, A.A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of variou
s h
uman 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
Bio
logy of The Cell., 3rd Ed., Garland
Publishing, New York and London, pp.


gene

gene

gene

gene

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

7

Definition and useful of transgenic mice


The useful of transgenic mice
2,3,10


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

_____________________________________________________________________________________________________________________________
___
__________________
___

2
Jusuf, A.A : Transgenic and gene disruption techniques from a concept to a tool in studying the basic pathogenesis of variou
s h
uman 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

ge
ne into eggs. Cell 1981: 27:
223
-
231

10
Nagy A, Gertsenstein M, Vintersen K, Behringer R, Manipulating the mouse embryo: A laboratory manual. 3rd Ed New York: Cold S
pri
ng



Harbor: 2003


Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

8

The procedures of producing transgenic mice


The processes of making transgenic mice


difficult and complicated


take time, energy and high cost


2 methods
19,20


Pronuclear microinjection


Embryonic Stem (ES) cell electroporation and
subsequent blastocyst injection

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

9

Method of making transgenic mice

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

10

The procedures of producing transgenic mice


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

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


DNA fragment or transgene


promoter,
complete protein coding region
, < satu intron dan
polyadenylation site
11



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 vector
12


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
. A
cad.Sci.USA 1988: 85; 836
-
40

12
Chada

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 dep
osi
tion 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



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

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


Endogenous transgene


insertion of oligonucleotide in the untranslated region of DNA fragment
14,15


insertion the small gene that express small RNA in transgene
16


fusion of protein transgen with protein Green Fluorescent Protein (GFP)
encoded by gene reporter alkaline phosphatase
17


______________________________________________________________________

14
Peschon J.J, Behringer R.R, Brinster R.L, and Palmiter R.D. Spermatide
-
specific expression of protamine 1 in transgenic mice. Pr
oc 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 famil
y.

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

14

The procedures of producing transgenic mice


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


Efficiency of transgene integration
8,10



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.

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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



Spring

Harbor
:

2003


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


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

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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

The procedures of producing transgenic mice


Preparation of Mice
8


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

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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

17

Vasectomization in the Sterile Male Mice


Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

18

The procedures of producing transgenic mice


Preparation of Mice
8


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

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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


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

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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


Holding pipette


Borosilicate glass capilary


mechanical pipette puller


Injection pipette


Glass capillary tubing dengan
internal glass filament


mechanical pipette puller

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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

The procedures of producing transgenic mice


Preparation of tools and materials
8


Microscope with micromanipulator


Paraffin oil


Petri dish injection chamber


Transgen


Detection of transgene


Southern blot


Northern blot


Western Blot

______________________________________________________________________

8
Hogan

B,

ConstantiniF,

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

22

Steps in Production of transgenic mice

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

23

Steps in Production of transgenic mice


Injection of hormone PMS and hHCG

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


Isolation and collecting 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


Injection of
transgene into male
pronuclear of
fertilized eggs

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


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

40

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

41

Steps in Production of transgenic mice


Detection of
Transgen
e

in the candidate of transgenic mice

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


Patogenesis and patophysiology of cancer


Regulation of genes belongs to oncogene and tumor
suppressor


Abnormality of tissue

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


Green et.al
21



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 <<

_____________________________________________________________________

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

44

The using of transgenic mice in studying the
carcinogenesis


Transgenic mice


Janz et.al
22



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

______________________________________________________________________

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 mammar
y t
issue.
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

45

The using of transgenic mice in studying the
carcinogenesis


Transgenic mice


Kim et.al.
24


i
nsertion entire
HBx

gene under its own regulatory elements


t
o study the role of that gene in patogenesis of hepatoma


r
esults


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

46

The using of transgenic mice in studying the
carcinogenesis


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


Arbeit et.al
2
5


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.


_____________________________________________________________________

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

47

The using of transgenic mice in studying the
carcinogenesis


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


Riley et.al.
26


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 E6 and E7 Func
tio
n in
Transgenic Mouse Models of Cervical Carcinogenesis. Cancer Research.2003
:

63:4862
-
4871

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

48

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.

_____________________________________________________________________

26
Riley, R.R., Duensing,S., Brake,T., Münger,K., Lambert, P.F., Arbeit, J.M. Dissection of Human Papillomavirus E6 and E7 Func
tio
n in
Transgenic Mouse Models of Cervical Carcinogenesis. Cancer Research.2003
:

63:4862
-
4871

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
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49

The using of transgenic mice in studying the
carcinogenesis


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

_____________________________________________________________________

27
Searle, P.F., Thomas, D.P., Faulkner, K.B., Tinsley, J.M. Stomach Cancer in Transgenic Mice Expressing Human Papillomavirus T
ype

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

50

The using of transgenic mice in studying the
carcinogenesis


Transgenic mice


Venkateswaran et.al
28



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

_____________________________________________________________________

28
Venkateswaran, V., Fleshner, N.E., Sugar,L.M., Klozt, L.H. Antioxidants block prostate cancer in lady transgenic mice. Cancer

Re
search
.2004:64:5891
-
96.

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
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FKUI/2009

51

The using of transgenic mice in studying the
carcinogenesis


Transgenic mice


Kato et.al
29



metallothionein
-
I (MT)/
ret

transgenic mouse and melanoma cell (Mel
-
ret)

transgenic mice


h
erbal
m
edicine
s
ho
-
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

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Establishing of Animal Model
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52

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 a
nimal 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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(transgenic mice)/AAJ/Histo
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FKUI/2009

53

Wednesday, Agust
12, 009

Establishing of Animal Model
(transgenic mice)/AAJ/Histo
-
FKUI/2009

54