CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC MOUSE MODEL FOR FRAGILE X-ASSOCIATED TREMOR ATAXIA SYNDROME (FXTAS) - Diponegoro University | Institutional Repository (UNDIP-IR)

CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC
MOUSE MODEL FOR FRAGILE X-ASSOCIATED
TREMOR/ATAXIA SYNDROME (FXTAS)

THESIS
A thesis submitted for the degree of Master of Biomedical Science majoring
on Genetic Counseling

by
FATWA ADIKUSUMA
G4A008039

POST GRADUATE PROGRAM
DIPONEGORO UNIVERSITY
SEMARANG
2011

CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC
MOUSE MODEL FOR FRAGILE X-ASSOCIATED
TREMOR/ATAXIA SYNDROME (FXTAS)

THESIS
A thesis submitted for the degree of Master of Biomedical Science majoring
on Genetic Counseling

by
FATWA ADIKUSUMA
G4A008039

POST GRADUATE PROGRAM
DIPONEGORO UNIVERSITY
SEMARANG
2011

THESIS
CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC
MOUSE MODEL FOR FRAGILE X-ASSOCIATED
TREMOR/ATAXIA SYNDROME (FXTAS)
By
FATWA ADIKUSUMA
G4A008039

Has been defended in front of the defense committee
On April 13th, 2011
and has been approved by:

The Netherland
Principal Supervisor,

Indonesia
Principal Supervisor,

Renate Hukema, PhD

Prof. Sultana MH Faradz, PhD
NIP. 130 701 415

Supervisor,

Supervisor,

Rob Willemsen, PhD

Prof. DR. dr. Sarjadi, Sp.PA (K)
NIP. 130 352 547
Recognition,

Science
Post Graduate Program Diponegoro University

DR. dr. Winarto Sp.MK, SpM(K)
NIP. 130 675 157

ii

DECLARATION

I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by
another person nor material which to a substantial extent has been accepted for the
award of any other degree or diploma of the university or other institute of higher

learning, except where due acknowledgement is made in the text.

Semarang, April 2011

Fatwa Adikusuma

iv

ACKNOWLEDGMENT
Thanks God for Your destiny to give me an amazing study experience in
this program, Genetic Counseling Biomedical Science Diponegoro University. In
this program many people and parties have contributed a lot for my study. I would
like to thank to them.
I would like to express my gratitude to my supervisors: Renate Hukema,
PhD, Rob Willemsen, PhD,

Professor Sultana MH Faradz MD, PhD, and

Professor Dr. dr. Sarjadi, SpPA (K) for the supervision, advices and support.
My special thanks to Professor dr. Sultana M.H. Faradz, PhD. Head of

Master Biomedical Science Majoring on Genetic Counseling Diponegoro
University Semarang for making this study happen and possible. Every single step
of this study has your contribution. Thank you for opening this program,
encouraging us to have better knowledge and experience, for your care to us, for
finding the scholarship, for supervising me, for everything tha
entirely.

dr. Tri Indah Winarni, MSi.Med for

her support to this program as well.
I also thank to Dr. Abe Susanto, MSc from from Bureau of Planning and
International Cooperation, Ministry of National Education The republic Indonesia,
who
I would like to thanks to Professor dr. Ben Oostra PhD and Rob
Willemsen PhD, from Department of Clinical Genetics, Erasmus MC Rotterdam,
The Netherlands, for making it possible to study and experience an advanced
research at their laboratory.

v

I would like to thank to colleagues in Fragile X group Erasmus MC who
gave many contributions whilst I was in the laboratory. Thanks to Edwin Mientjes
PhD, Liesanne and Ronald Buijsen.
I would like to extend my acknowledgement to dr. Endang
Ambarwati,Sp.RM, dean of the Faculty of Medicine Diponegoro University
Semarang, and also to
Program in Biomedical Science Post Graduate Program Diponegoro University
Semarang, Faculty of Medicine, Diponegoro University Semarang.
Last but not least, I would like to thank to my family and many people
who contribute in supporting me to finish this study.

Semarang, April 2011

Fatwa Adikusuma

vi

LIST OF ABBREVIATIONS

ASO

: antisense oligonucleotides

(CGG)n

: CGG with (n) number of repeat

C57BL/6

: a type of mouse strain which is widely used for
laboratory mouse

COS-7

: CV-1 (simian) in Origin, and carrying the SV40
genetic material line 7

DM

: myotonic dystrophy

DMPK

: myotonic dystrophiy protein kinase

dox

: doxycyline

eGFP

: enhanced green fluorescent protein

FISH

: fluorescence in situ hybridization

Fmr1

: fragile x mental retardation 1 (for animal)

FMR1

: fragile x mental retardation 1 (for human)

Fmrp

: fragile x mental retardation protein (for animal)

FMRP

: fragile x mental retardation protein (for human)

FXTAS

: fragile x-associated tremor/ataxia syndrome

Gapdh

: glyceraldehyde 3-phosphate dehydrogenase

GFA

: glial fibrillary acidic

GFP

: green fluorescent protein

GLAST

: glutamate aspartate transporters

hnRNP

: Heterogeneous nuclear ribonucleoproteins

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Hsp

: heat shock protein

IP

: intraperitoneal

KI

: knock-in

MBNL1

: muscleblind-like 1

MCP

: middle cerebellar peduncle

MRI

: magnetic resonance imaging

Pcp

: Purkinje cell protein

POF

: premature ovarian failure

PrP

: prion protein

RT-Q-PCR

: reverse transcriptase-quantitative-polymerase
chain reaction

rtTA

: reverse-tetracycline transactivator

SCA

: spinocerebellar ataxia

Tet

: tetracycline

UTR

: untranslated region

ZNF9

: zinc finger protein 9

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TABLE OF CONTENTS
DECLARATION ................................................................................................................iv
ACKNOWLEDGMENT ..................................................................................................... v
LIST OF ABBREVIATIONS ............................................................................................ vii
TABLE OF CONTENTS ....................................................................................................ix
LIST OF TABLE, FIGURES, AND APPENDICES ..........................................................xi
ABSTRACT ...................................................................................................................... xiii
ABSTRAK ........................................................................................................................ xiv
CHAPTER 1
INTRODUCTION .............................................................................................................. 1
1.1. BACKGROUND ..................................................................................................... 1
1.2. RESEARCH QUESTIONS...................................................................................... 5
1.3. RESEARCH PURPOSES ........................................................................................ 6
1.4. RESEARCH BENEFITS ......................................................................................... 7
1.5. RESEARCH ORIGINALITY .................................................................................. 7
CHAPTER 2
LITERATURE REVIEW ................................................................................................... 8
2.1. FXTAS: THE FMR1 GENE AND CLINICAL ASPECTS ..................................... 8
2.2. NEUROPATHOLOGICAL OF FXTAS ............................................................... 11
2.3. MOLECULAR PATHOGENESIS OF FXTAS .................................................... 14
2.4. MOUSE MODEL OF FXTAS............................................................................... 17
CHAPTER 3
THEORITICAL FRAMEWORK, CONCEPTUAL FRAMEWORK, AND
HYPOTHESES ................................................................................................................. 20
3.1. THEORITICAL FRAMEWORK .......................................................................... 20
3.2. CONCEPTUAL FRAMEWORK .......................................................................... 21
3.3. HYPOTHESES ...................................................................................................... 22
CHAPTER 4
RESEARCH METHODS ................................................................................................. 23
4.1. RESEARCH FIELD .............................................................................................. 23
4.2. SETTING/LOCATION, RESEARCH PERIOD ................................................... 23

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4.3. RESEARCH DESIGN ........................................................................................... 23
4.4. POPULATIONS AND SAMPLES........................................................................ 23
4.5. RESEARCH VARIABLES ................................................................................... 24
4.6. OPERATIONAL DEFINITIONS .......................................................................... 25
4.7. RESEARCH PROTOCOLS .................................................................................. 27
4.8. FRAMEWORK OF RESEARCH .......................................................................... 36
4.9. DATA ANALYSIS ................................................................................................ 44
4.10. ETHICAL IMPLICATION.................................................................................. 44
CHAPTER 5
RESULTS ......................................................................................................................... 45
5.1. Previous study: cell culture and organotypical slices ............................................ 45
5.2. The workable of Tet-on-nCGG-eGFP: in vivo study using Tet-on-nCGGeGFP/hnRNP-rtTA ....................................................................................................... 45
5.3. The workable of Tet-on-99CGG-eGFP/GFA2-rtTA in vivo ................................. 48
5.4. The workable of Tet-on-99CGG-eGFP/PrP-rtTA in vivo ...................................... 48
5.5. Transgene expression outside the brain in Tet-on-99CGG-eGFP/PrP-rtTA bigenic
mice ............................................................................................................................... 50
5.6. Best founder of PrP-rtTA ....................................................................................... 53
5.7. Best founder of Tet-on-99CGG-eGFP ................................................................... 54
5.8. Formation of ubiquitin intranuclear inclusion in the Tet-on-99CGG-eGFP/PrPrtTA bigenic mice ......................................................................................................... 57
CHAPTER 6
DISCUSSION ................................................................................................................... 62
6.1. Leakage of Tet-on-99CGG-eGFP/PrP-rtTA .......................................................... 63
6.2. Toxic RNA and inclusions ..................................................................................... 64
6.3. Reversibility study ................................................................................................. 66
6.4. Astrocytes and glutamate excitotoxicity involvement for disease progression ..... 67
CHAPTER 7
CONCLUSION AND SUGGESTION ............................................................................. 72
REFERENCES ................................................................................................................. 73
CURRICULUM VITAE ................................................................................................... 81
APPENDICES .................................................................................................................. 85

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LIST OF TABLE, FIGURES, AND APPENDICES

Tables
Table 1. Proteins found in the human brain inclusions

Figures
Figure 1. CGG length, FMR1 expression and the clinical outcomes
Figure 2. Ubiquitin-positive intranuclear inclusions in human neurons, human
astrocytes, murine neurons
Figure 3. Immunohistochemistry for GFP in liver, kidney, and intestine of
bigenic mice Tet-on-nCGG-eGFP/hnRNP-rtTA with dox treatment
Figure 4. Immunohistochemistry for GFP in brain slices of bigenic mice Tet-on99CGG-eGFP/PrP-rtTA after 2 weeks of dox in drinking treatment
Figure 5. Immunohistochemistry for GFP in tissues outside the brain of bigenic
mice Tet-on-99CGG-eGFP/PrP-rtTA after dox intraperitoneal injection
Figure 6. Western blot for GFP on kidney of tet-on-99CGG-eGFP/PrP-rtTA
mouse after dox IP treatment
Figure 7. RT-Q-PCR for GFP RNA from kidney of tet-on-99CGG-eGFP/PrPrtTA mouse after dox IP treatment
Figure 8. rtTA mRNA levels from the brains of different PrP-rtTA founder
Figure 9. Immunohistochemistry for GFP in the liver slices comparing founder
31 and 39 of Tet-on-99CGG-eGFP
Figure 10. GFP RNA levels in the liver comparing founder 31 and 39 of Tet-on99CGG-eGFP
Figure 11. Western blot analysis for GFP in the liver comparing founder 31 and
39 of Tet-on-99CGG-eGFP
Figure 12. Immunohistochemistry for GFP in the brain slices of bigenic mice Teton-99CGG-eGFP/PrP-rtTA after dox treatment

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Figure 13. GFP RNA levels in the brain of bigenic mice Tet-on-99CGGeGFP/PrP-rtTA after dox treatments
Figure 14. Western blot for GFP of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA
after different time points of dox treatment
Figure 15. Immunohistochemistry of ubiquitin in the brain slices of bigenic mice
Tet-on-99CGG-eGFP/PrP-rtTA with 12 and 16 weeks of dox treatment

Appendices
Appendix 1. rtTA RT-Q-PCR from brain result for choosing the best PrP-rtTA
founder
Appendix 2. Western blot for GFP in liver of mice from founder 39 vs 31 of Teton-99CGG-eGFP
Appendix 3. RT-Q-PCR for GFP in liver of mice from founder 39 vs 31 of Teton-99CGG-eGFPA
Appendix 4. RT-Q-PCR for GFP after different time points of dox treatment
Appendix 5. Western blot for GFP after different time points of dox treatment
Appendix 6. Ethical clearance document

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ABSTRACT
Background: FXTAS (fragile X-associated tremor/ataxia syndrome) is a
neurodegenerative disease with tremor and ataxia as the primary symptoms and
ubiquitin-positive intranuclear inclusions as the major neuropathological
hallmark. FXTAS affects premutation males with an expanded premutation CGG
repeat in the promoter of the FMR1 gene. The expanded premutation CGG RNA
is considered toxic. Inducible transgenic mice were generated, using the Tet-on
system to express transgene expanded premutation 99CGG along with an eGFP
reporter under control of dox (doxycycline) induction, as well as specific driver
promoter, the GFA2-rtTA and PrP-rtTA. This study aims to characterize those
mouse models so they would be ready to be used for further studies.
Methods: Immunohistochemistry, Western blot, and RT-Q-PCR techniques were
used to characterize the transgene expression of the transgenic mice.
Characterization of ubiquitin inclusions formation used immunohistochemistry
technique.
Results: The Tet-on-99CGG-eGFP transgene worked in vivo, while Tet-on11CGG-eGFP lost its expression. Tet-on-99CGG-eGFP/GFA2-rtTA bigenic mice
did not work in vivo, while Tet-on-99CGG-eGFP/PrP-rtTA bigenic mice did
work. As expected, the Tet-on-99CGG-eGFP/PrP-rtTA mice formed the FXTAS
hallmark, the ubiquitin-positive intranuclear inclusions after 12 weeks of dox
induction.
Conclusions: Mouse model Tet-on-99CGG-eGFP/PrP-rtTA worked well, and
were ready to be used for main further studies.

Keywords: FXTAS, FMR1 gene, transgenic mice, Tet-on, PrP, GFA2,
reversibility, ubiquitin inclusions

xiii

ABSTRAK
Latar belakang: FXTAS (fragile X-associated tremor/ataxia syndrome)
merupakan penyakit neurodegeneratif dengan gejala utama tremor dan ataxia serta
inklusi intranuklear ubiquitin yang muncul sebagai ciri-ciri utama secara
neuropatologikal. FXTAS menyerang pria premutasi yang mengalami ekspansi
repetasi CGG di promotor gen FMR1. RNA CGG premutasi dipercaya toksik.
Tikus transgenik yang dapat diinduksi sudah dibuat, dengan menggunakan sistem
Tet-on untuk mengekspresikan transgen 99CGG terekspansi bersama-sama
dengan reporter eGFP, serta dapat dikontrol melalui induksi dox (doxycycline),
juga menggunakan promotor pengendali yang spesifik yaitu GFA2-rtTA dan
PrP-rtTA. Studi ini bertujuan untuk mengkarakterisasi tikus tersebut sehingga
model tikus tersebut siap untuk digunakan untuk studi-studi berikutnya.
Metode: Teknik imunohistokimia, Western blot, dan RT-Q-PCR digunakan untuk
mengkarakterisasi ekspresi transgene pada tikus transgenik. Karakterisasi inklusi
ubiquitin menggunakan teknik imunohistokimia.
Hasil: Transgen Tet-on-99CGG-eGFP dapat bekerja secara in vivo, tetapi Tet-on11CGG-eGFP kehilangan ekspresi transgennya. Tikus bigenik Tet-on-99CGGeGFP/GFA2-rtTA tidak bekerja, sedangkan tikus bigenik Tet-on-99CGGeGFP/PrP-rtTA dapat bekerja dengan baik. Sesuai harapan, tikus bigenik Tet-on99CGG-eGFP/PrP-rtTA dapat menghasilkan inklusi intranuklear ubiquitin setelah
induksi dox selama 12 minggu.
Kesimpulan: Model tikus Tet-on-99CGG-eGFP/PrP-rtTA dapat bekerja dengan
baik dan siap untuk digunakan untuk studi penting berikutnya

Kata kunci: FXTAS, gen FMR1, tikus transgenik, Tet-on, PrP, GFA2,
reversibilitas, inklusi ubiquitin

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