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Generation of transgenic mice to evaluate promoter activity and specificity of two human endogenous retrovirus long terminal repeats [Elektronische Ressource] / by Regine Margarete Schönfeld

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Institute of Molecular Animal Breeding and Biotechnology, Gene Center Institute of Molecular Animal Breeding and Biotechnology, Gene Center Faculty of Veterinary Medicine of the Ludwig-Maximilians-University Munich Faculty of Veterinary Medicine of the Ludwig-Maximilians-University Munich Prof. Dr. Eckhard Wolf Prof. Dr. Eckhard Wolf Generation of Transgenic Mice to Evaluate Promoter Activity and Generation of Transgenic Mice to Evaluate Promoter Activity and Specificity of Two Human Endogenous Retrovirus Specificity of Two Human Endogenous Retrovirus Long Terminal Repeats Long Terminal Repeats Thesis for the attainment of the title Doctor in Veterinary Medicine Thesis for the attainment of the title Doctor in Veterinary Medicine from the Faculty of Veterinary Medicine of the Ludwig-Maximilians-University, Munich from the Faculty of Veterinary Medicine of the Ludwig-Maximilians-University, Munich By By Regine Margarete Schönfeld Regine Margarete Schönfeld from Cologne from Cologne Munich 2003 Munich 2003 Contents II Contents IIAus dem Institut für Tierzucht der Tierärztlichen Fakultät der Universität München Aus dem Institut für Tierzucht der Tierärztlichen Fakultät der Universität München Lehrstuhl für Molekulare Tierzucht und Biotechnologie Lehrstuhl für Molekulare Tierzucht und Biotechnologie Prof. Dr. Eckhard Wolf Prof. Dr.

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Published 01 January 2003
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Institute of Molecular Animal Breeding and Biotechnology, Gene Center Institute of Molecular Animal Breeding and Biotechnology, Gene Center
Faculty of Veterinary Medicine of the Ludwig-Maximilians-University Munich Faculty of Veterinary Medicine of the Ludwig-Maximilians-University Munich
Prof. Dr. Eckhard Wolf Prof. Dr. Eckhard Wolf








Generation of Transgenic Mice to Evaluate Promoter Activity and Generation of Transgenic Mice to Evaluate Promoter Activity and
Specificity of Two Human Endogenous Retrovirus Specificity of Two Human Endogenous Retrovirus
Long Terminal Repeats Long Terminal Repeats




Thesis for the attainment of the title Doctor in Veterinary Medicine Thesis for the attainment of the title Doctor in Veterinary Medicine
from the Faculty of Veterinary Medicine of the Ludwig-Maximilians-University, Munich from the Faculty of Veterinary Medicine of the Ludwig-Maximilians-University, Munich






By By
Regine Margarete Schönfeld Regine Margarete Schönfeld
from Cologne from Cologne

Munich 2003 Munich 2003 Contents II Contents II
Aus dem Institut für Tierzucht der Tierärztlichen Fakultät der Universität München Aus dem Institut für Tierzucht der Tierärztlichen Fakultät der Universität München
Lehrstuhl für Molekulare Tierzucht und Biotechnologie Lehrstuhl für Molekulare Tierzucht und Biotechnologie
Prof. Dr. Eckhard Wolf Prof. Dr. Eckhard Wolf








Untersuchungen zur Promotor-Aktivität und -Spezifität von zwei Untersuchungen zur Promotor-Aktivität und -Spezifität von zwei
Long Terminal Repeats humaner endogener Retroviren Long Terminal Repeats humaner endogener Retroviren
in transgenen Mäusen in transgenen Mäusen




Inaugural-Dissertation Inaugural-Dissertation
zur Erlangung der tiermedizinischen Doktorwürde zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München der Ludwig-Maximilians-Universität München



Von Von
Regine Margarete Schönfeld Regine Margarete Schönfeld
aus Köln aus Köln

München 2003 München 2003 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Gedruckt mit Genehmigung der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München Ludwig-Maximilians-Universität München









Dekan: Univ.-Prof. Dr. R. Stolla Dekan: Univ.-Prof. Dr. R. Stolla

Referent: Univ.-Prof. Dr. E. Wolf Referent: Univ.-Prof. Dr. E. Wolf

Korreferent: Priv.-Doz. Dr. Dr. R. G. Erben Korreferent: Priv.-Doz. Dr. Dr. R. G. Erben










Tag der Promotion: 7. Februar 2003 Tag der Promotion: 7. Februar 2003


















Meiner Familie Meiner Familie I Contents I Contents
CONTENTS CONTENTS

1 INTRODUCTION AND OBJECTIVES............................................................................. 1 1 INTRODUCTION AND OBJECTIVES............................................................................. 1
2 REVIEW OFTHE LITERATURE .................................................................................... 2 2 REVIEW OFTHE LITERATURE .................................................................................... 2
2.1 Genetic engineering of the mouse........................................................................................ 2 2.1 Genetic engineering of the mouse........................................................................................ 2
2.1.1 Transgenic technology............................................................................................................ 2 2.1.1 Transgenic technology............................................................................................................ 2
2.1.2 Mouse models in genetics....................................................................................................... 3 2.1.2 Mouse models in genetics....................................................................................................... 3
2.1.3 Transgenic mice...................................................................................................................... 5 2.1.3 Transgenic mice...................................................................................................................... 5
2.1.4 Design of the gene construct................................................................................................... 6 2.1.4 Design of the gene construct................................................................................................... 6
2.2 Human endogenous retroviruses......................................................................................... 7 2.2 Human endogenous retroviruses......................................................................................... 7
2.2.1 Characteristics and biological significance of human endogenous retroviruses..................... 7 2.2.1 Characteristics and biological significance of human endogenous retroviruses..................... 7
2.2.2 In vitro and in vivo models for testing promoter activity of HERV-LTRs ........................... 10 2.2.2 In vitro and in vivo models for testing promoter activity of HERV-LTRs ........................... 10
2.2.3 Characteristics and biological significance of the HERV-H-H6 LTR.................................. 12 2.2.3 Characteristics and biological significance of the HERV-H-H6 LTR.................................. 12
2.2.4 Characteristics and significance of the HERV-L LTR......................................... 13 2.2.4 Characteristics and significance of the HERV-L LTR......................................... 13
2.2.5 Potential use of HERV-LTRs in gene therapy...................................................................... 15 2.2.5 Potential use of HERV-LTRs in gene therapy...................................................................... 15
2.3 The use of Green Fluorescent Protein (GFP) as reporter gene....................................... 17 2.3 The use of Green Fluorescent Protein (GFP) as reporter gene....................................... 17
2.3.1 The discovery of GFP........................................................................................................... 18 2.3.1 The discovery of GFP........................................................................................................... 18
2.3.2 Structure of GFP and Enhanced Green Fluorescent Protein (EGFP).................................... 18 2.3.2 Structure of GFP and Enhanced Green Fluorescent Protein (EGFP).................................... 18
2.3.3 Biochemical and physical properties of EGFP ..................................................................... 20 2.3.3 Biochemical and physical properties of EGFP ..................................................................... 20
2.3.4 Application of GFPs as reporter gene................................................................................... 22 2.3.4 Application of GFPs as reporter gene................................................................................... 22
2.3.5 Applications of EGFP in different species............................................................................ 23 2.3.5 Applications of EGFP in different species............................................................................ 23
2.3.5 Sensitivity of GFP and EGFP as reporter gene 24 2.3.5 Sensitivity of GFP and EGFP as reporter gene 24
2.3.6 Qualitative and quantitative analysis of GFP and EGFP expression in transgenic mice ...... 25 2.3.6 Qualitative and quantitative analysis of GFP and EGFP expression in transgenic mice ...... 25
2.4 The use of Firefly Luciferase as reporter gene in transgenic mice................................. 29 2.4 The use of Firefly Luciferase as reporter gene in transgenic mice................................. 29
2.4.1 Structure and characteristics of firefly luciferase ................................................................. 29 2.4.1 Structure and characteristics of firefly luciferase ................................................................. 29
2.4.2 Qualitative and quantitative analysis of firefly luciferase in transgenic mice ...................... 30 2.4.2 Qualitative and quantitative analysis of firefly luciferase in transgenic mice ...................... 30
3 ANIMALS, MATERIALS AND METHODS .................................................................. 35 3 ANIMALS, MATERIALS AND METHODS .................................................................. 35
3.1 Animals................................................................................................................................ 35 3.1 Animals................................................................................................................................ 35
3.1.1 Mice...................................................................................................................................... 35 3.1.1 Mice...................................................................................................................................... 35
3.1.2 Housing and husbandry ........................................................................................................ 35 3.1.2 Housing and husbandry ........................................................................................................ 35
3.1.3 Breeding system ................................................................................................................... 36 3.1.3 Breeding system ................................................................................................................... 36
3.2 Preparation of constructs for DNA-microinjection ......................................................... 36 3.2 Preparation of constructs for DNA-microinjection ......................................................... 36
3.2.1 The pBL-HERV-L construct................................................................................................36 3.2.1 The pBL-HERV-L construct................................................................................................36
3.2.2 The pEGFP-HERV-H-H6 construct.....................................................................................37 3.2.2 The pEGFP-HERV-H-H6 construct.....................................................................................37
3.2.3 Preparation of competent bacteria ........................................................................................3.2.3 Preparation of competent bacteria ........................................................................................Contents II Contents II
3.2.4 Transformation of bacteria................................................................................................... 38 3.2.4 Transformation of bacteria................................................................................................... 38
3.2.5 Preparation of plasmid DNA (Miniprep).............................................................................. 39 3.2.5 Preparation of plasmid DNA (Miniprep).............................................................................. 39
3.2.6 Restriction enzyme digestion................................................................................................ 40 3.2.6 Restriction enzyme digestion................................................................................................ 40
3.2.7 Extraction of DNA fragments from agarose gels.................................................................. 42 3.2.7 Extraction of DNA fragments from agarose gels.................................................................. 42
3.3 Production of transgenic mice ........................................................................................... 43 3.3 Production of transgenic mice ........................................................................................... 43
3.3.1 Superovulation and isolation of fertilized oocytes................................................................ 43 3.3.1 Superovulation and isolation of fertilized oocytes................................................................ 43
3.3.2 Microinjection ...................................................................................................................... 43 3.3.2 Microinjection ...................................................................................................................... 43
3.3.3 Transfer of embryos into the oviduct of synchronized recipients......................................... 44 3.3.3 Transfer of embryos into the oviduct of synchronized recipients......................................... 44
3.4 Identification of transgenic mice ....................................................................................... 44 3.4 Identification of transgenic mice ....................................................................................... 44
3.4.1 Identification using the Polymerase Chain Reaction (PCR)................................................. 44 3.4.1 Identification using the Polymerase Chain Reaction (PCR)................................................. 44
3.4.1.1 Preparation of genomic DNA from mouse tails............................................................... 44 3.4.1.1 Preparation of genomic DNA from mouse tails............................................................... 44
3.4.1.2 PCR conditions ................................................................................................................ 45 3.4.1.2 PCR conditions ................................................................................................................ 45
3.4.1.3 Agarose gel electrophoresis ............................................................................................. 47 3.4.1.3 Agarose gel electrophoresis ............................................................................................. 47
3.4.2 Southern blot analysis........................................................................................................... 47 3.4.2 Southern blot analysis........................................................................................................... 47
3.4.2.1 Extraction of genomic DNA and determination of concentration.................................... 47 3.4.2.1 Extraction of genomic DNA and determination of concentration.................................... 47
3.4.2.2 Digestion of genomic DNA ............................................................................................. 48 3.4.2.2 Digestion of genomic DNA ............................................................................................. 48
3.4.2.3 Transfer of the DNA ........................................................................................................ 49 3.4.2.3 Transfer of the DNA ........................................................................................................ 49
3.4.2.4 Radioactive probe labeling. 51 3.4.2.4 Radioactive probe labeling. 51
3.4.2.5 Hybridization, washing and signal detection ................................................................... 52 3.4.2.5 Hybridization, washing and signal detection ................................................................... 52
3.5 Evaluation of gene expression at RNA level ..................................................................... 52 3.5 Evaluation of gene expression at RNA level ..................................................................... 52
3.5.1 Reverse transcription PCR (RT-PCR) .................................................................................. 52 3.5.1 Reverse transcription PCR (RT-PCR) .................................................................................. 52
3.5.1.1 Extraction of RNA from mouse tissue............................................................................. 53 3.5.1.1 Extraction of RNA from mouse tissue............................................................................. 53
3.5.1.2 Reverse transcription of mRNA and PCR from cDNA ................................................... 54 3.5.1.2 Reverse transcription of mRNA and PCR from cDNA ................................................... 54
3.6 Evaluation of gene expression at the protein level........................................................... 54 3.6 Evaluation of gene expression at the protein level........................................................... 54
3.6.1 Western Blot......................................................................................................................... 54 3.6.1 Western Blot......................................................................................................................... 54
3.6.1.1 Extraction of protein from tissue samples........................................................................ 54 3.6.1.1 Extraction of protein from tissue samples........................................................................ 54
3.6.1.2 Determination of protein concentration........................................................................... 55 3.6.1.2 Determination of protein concentration........................................................................... 55
3.6.1.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .................................................. 55 3.6.1.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .................................................. 55
3.6.1.4 Electroblotting ................................................................................................................. 57 3.6.1.4 Electroblotting ................................................................................................................. 57
3.6.1.5 Protein detection by peroxidase-labeled antibodies......................................................... 57 3.6.1.5 Protein detection by peroxidase-labeled antibodies......................................................... 57
3.6.2 Histology.............................................................................................................................. 58 3.6.2 Histology.............................................................................................................................. 58
3.6.2.1 Collection and fixation of tissues..................................................................................... 58 3.6.2.1 Collection and fixation of tissues..................................................................................... 58
3.6.2.2 Preparation of slides......................................................................................................... 59 3.6.2.2 Preparation of slides......................................................................................................... 59
3.6.2.3 Haematoxylin & eosin staining........................................................................................ 60 3.6.2.3 Haematoxylin & eosin staining........................................................................................ 60
3.6.2.4 Mowiol and propidium iodide staining............................................................................ 60 3.6.2.4 Mowiol and propidium iodide staining............................................................................ 60
3.6.2.5 Immunohistochemical staining 60 3.6.2.5 Immunohistochemical staining 60
3.6.2.6 Histological investigation of tissues ................................................................................ 61 3.6.2.6 Histological investigation of tissues ................................................................................ 61 III Contents III Contents
3.7 Phenotypic consequences of transgene expression........................................................... 61 3.7 Phenotypic consequences of transgene expression........................................................... 61
3.7.1 Analysis of body weight, body length and organ weights .................................................... 61 3.7.1 Analysis of body weight, body length and organ weights .................................................... 61
3.8 Statistics............................................................................................................................... 62 3.8 Statistics............................................................................................................................... 62
3.9 Equipment and reagents .................................................................................................... 62 3.9 Equipment and reagents .................................................................................................... 62
4 RESULTS............................................................................................................................ 67 4 RESULTS............................................................................................................................ 67
4.1 Purification of gene constructs for DNA microinjection................................................. 67 4.1 Purification of gene constructs for DNA microinjection................................................. 67
4.2 Generation of transgenic animals and breeding of transgenic lines............................... 67 4.2 Generation of transgenic animals and breeding of transgenic lines............................... 67
4.3 Expression studies............................................................................................................... 72 4.3 Expression studies............................................................................................................... 72
5 DISCUSSION...................................................................................................................... 81 5 DISCUSSION...................................................................................................................... 81
5.1 Analysis of transgene integration ...................................................................................... 81 5.1 Analysis of transgene integration ...................................................................................... 81
5.2 Expression level and pattern in pBL-HERV-L transgenic mice .................................... 81 5.2 Expression level and pattern in pBL-HERV-L transgenic mice .................................... 81
5.3 on level and pattern in pEGFP-HERV-H6 transgenic mice............................. 82 5.3 on level and pattern in pEGFP-HERV-H6 transgenic mice............................. 82
5.4 Final considerations............................................................................................................ 86 5.4 Final considerations............................................................................................................ 86
6 SUMMARY......................................................................................................................... 89 6 SUMMARY......................................................................................................................... 89
7 ZUSAMMENFASSUNG....................................................................................................91 7 ZUSAMMENFASSUNG....................................................................................................91
8 BIBLIOGRAPHY............................................................................................................... 93 8 BIBLIOGRAPHY............................................................................................................... 93
Abbreviations IV Abbreviations IV
ABBREVIATIONS ABBREVIATIONS

AIDS aquired immune deficiency syndrome AIDS aquired immune deficiency syndrome
AMP adenosine monophosphate AMP adenosine monophosphate
ATP adenosine triphosphate ATP adenosine triphosphate
BAC bacterial artificial chromosome BAC bacterial artificial chromosome
BCA bichioninic acid BCA bichioninic acid
bp base pair bp base pair
BSA bovine serum albumin BSA bovine serum albumin
CAT chloramphenicol acetyltransferase CAT chloramphenicol acetyltransferase
CCLR cell culture lysis buffer CCLR cell culture lysis buffer
cDNA complementary DNA cDNA complementary DNA
Chang liver human liver cell line Chang liver human liver cell line
CMV cytomegalovirus CMV cytomegalovirus
cm centimeter cm centimeter
cpm counts per minute cpm counts per minute
DAB 3,3´-diaminobenzidine DAB 3,3´-diaminobenzidine
DEPC diethylpyrocarbonate DEPC diethylpyrocarbonate
DMSO dimethyl sulfoxide DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid DNA deoxyribonucleic acid
dNTPs deoxynucleotide-tri-phosphates dNTPs deoxynucleotide-tri-phosphates
DQB class II gene of the major histocompatibility complex DQB class II gene of the major histocompatibility complex
DTNB 5,5´-dithio-bis-[2-nitrobenzoic acid] DTNB 5,5´-dithio-bis-[2-nitrobenzoic acid]
DTT dithiothreitol DTT dithiothreitol
EBFP enhanced blue fluorescent protein EBFP enhanced blue fluorescent protein
EBV Epstein-Barr virus EBV Epstein-Barr virus
ECL enhanced chemiluminescent system ECL enhanced chemiluminescent system
ECFP enhanced cyan fluorescent protein ECFP enhanced cyan fluorescent protein
E. coli Escherichia coli E. coli Escherichia coli
EGFP enhanced green fluorescent protein EGFP enhanced green fluorescent protein
EMBL european molecular biology lab EMBL european molecular biology lab
ES embryonic stem cells ES embryonic stem cells
EtBr ethidiumbromide EtBr ethidiumbromide
EYFP enhanced yellow fluorescent protein EYFP enhanced yellow fluorescent protein
FACS fluorescence-activated cell sorting FACS fluorescence-activated cell sorting
FCS fetal calf serum FCS fetal calf serum
FITC fluorescein isothiocyanate FITC fluorescein isothiocyanate
FRET fluorescence resonance energy transfer FRET fluorescence resonance energy transfer
g gram g gram
g gravity g gravity
GFP green fluorescent protein GFP green fluorescent protein
h hour h hour
HaCaT human keratinocyte cell line HaCaT human keratinocyte cell line
HC high concentrated HC high concentrated
HCG human chorionic gonadotropin HCG human chorionic gonadotropin
Hela human cervical adenocarcinoma cell line Hela human cervical adenocarcinoma cell line V Abbreviations V Abbreviations
Hep2 human adenocarcinoma cell line Hep2 human adenocarcinoma cell line
HERV human endogenous retrovirus HERV human endogenous retrovirus
HIV human immunodeficiency virus HIV human immunodeficiency virus
Huh-7 human liver cell line Huh-7 human liver cell line
ICCD intensified charged coupled device ICCD intensified charged coupled device
ICOS inducible T-cell co-stimulator ICOS inducible T-cell co-stimulator
IDDM insulin-dependent diabetes mellitus IDDM insulin-dependent diabetes mellitus
IMR induced mutant resource IMR induced mutant resource
kb kilobases kb kilobases
kDa kilo Dalton kDa kilo Dalton
LB medium Luria Bertani medium LB medium Luria Bertani medium
LC5 human lung fibroblast cell line LC5 human lung fibroblast cell line
LC-5 human lung carcinoma cell line LC-5 human lung carcinoma cell line
LTR long terminal repeat LTR long terminal repeat
MAR matrix attachment region MAR matrix attachment region
Mb megabases Mb megabases
MCF7 human mammary adenocarcinoma cell line MCF7 human mammary adenocarcinoma cell line
mg milligram mg milligram
MiaPaCa2 human pancreas carcinoma cell line MiaPaCa2 human pancreas carcinoma cell line
min minute min minute
ml milliliter ml milliliter
mm millimeter mm millimeter
mMillimolar mMillimolar
msilliseconds msilliseconds
M-MLV moloney murine leukemia virus M-MLV moloney murine leukemia virus
NADH nicotinamide-adenine dinucleotide (reduced form) NADH nicotinamide-adenine dinucleotide (reduced form)
ng nanogramng nanogram
nm nanometer nm nanometer
No. number No. number
NRL nose rump length NRL nose rump length
NT nuclear transfer NT nuclear transfer
NTera2D1 human teratocarcinoma cell line NTera2D1 human teratocarcinoma cell line
OD optical density OD optical density
PAC P1-derived artificial chromosomes PAC P1-derived artificial chromosomes
PBS phosphate buffered saline PBS phosphate buffered saline
PCR polymerase chain reaction PCR polymerase chain reaction
pH negative logarithm of the hydrogen ion concentration pH negative logarithm of the hydrogen ion concentration
PI propidium iodide PI propidium iodide
PLB passive lysis buffer PLB passive lysis buffer
PMSG pregnant mares serum gonadotropin PMSG pregnant mares serum gonadotropin
PP inorganic pyrophosphate PP inorganic pyrophosphate i i
Prnp prion protein Prnp prion protein
PVDF polyvinylidendiflouride PVDF polyvinylidendiflouride
RFP red fluorescent protein RFP red fluorescent protein
RLU relative light units RLU relative light units
RNA ribonucleic acid RNA ribonucleic acid