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Gene deletion and functional analysis of Fetuin-B [Elektronische Ressource] / vorgelegt von Jennifer Wessling

147 pages
"Gene Deletion and Functional Analysis of Fetuin-B" Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologin Jennifer Wessling geb. Goldstein aus Aachen Berichter: Universitätsprofessor Dr. rer. nat. W. Jahnen-Dechent Universitätsprofessor Dr. rer. nat. L. Elling Tag der mündlichen Prüfung: 21. November 2007 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Contents1 Introduction 51.1 Fetuin Family of Proteins . . . . . . . . . . . . . . . . . . . . 51.1.1 Protein Structure . . . . . . . . . . . . . . . . . . . . . 61.1.2 Expression . . . . . . . . . . . . . . . . . . . . . . . . . 101.1.3 Genetic Organization . . . . . . . . . . . . . . . . . . . 111.1.4 Biological Role . . . . . . . . . . . . . . . . . . . . . . 121.2 Female Reproduction . . . . . . . . . . . . . . . . . . . . . . . 141.2.1 Oogenesis and Ovulation . . . . . . . . . . . . . . . . . 141.2.2 Fertilization . . . . . . . . . . . . . . . . . . . . . . . . 161.2.3 Parthenogenesis . . . . . . . . . . . . . . . . . . . . . . 171.3 The Mouse as a Model System . . . . . . . . . . . . . . . . . . 171.3.1 Targeting the Mouse Genome . . . . . . . . . . . . . . 181.4 Aim of the Study . . . . . . . . . . . . . . . . . . . . . . . . .
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"Gene Deletion and Functional Analysis of Fetuin-B"

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades einer
Doktorin der Naturwissenschaften
genehmigte Dissertation

vorgelegt von
Diplom-Biologin
Jennifer Wessling geb. Goldstein
aus Aachen

Berichter: Universitätsprofessor Dr. rer. nat. W. Jahnen-Dechent
Universitätsprofessor Dr. rer. nat. L. Elling

Tag der mündlichen Prüfung: 21. November 2007


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Contents
1 Introduction 5
1.1 Fetuin Family of Proteins . . . . . . . . . . . . . . . . . . . . 5
1.1.1 Protein Structure . . . . . . . . . . . . . . . . . . . . . 6
1.1.2 Expression . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.1.3 Genetic Organization . . . . . . . . . . . . . . . . . . . 11
1.1.4 Biological Role . . . . . . . . . . . . . . . . . . . . . . 12
1.2 Female Reproduction . . . . . . . . . . . . . . . . . . . . . . . 14
1.2.1 Oogenesis and Ovulation . . . . . . . . . . . . . . . . . 14
1.2.2 Fertilization . . . . . . . . . . . . . . . . . . . . . . . . 16
1.2.3 Parthenogenesis . . . . . . . . . . . . . . . . . . . . . . 17
1.3 The Mouse as a Model System . . . . . . . . . . . . . . . . . . 17
1.3.1 Targeting the Mouse Genome . . . . . . . . . . . . . . 18
1.4 Aim of the Study . . . . . . . . . . . . . . . . . . . . . . . . . 20
2 Materials and Methods 21
2.1 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2 Generating a fetuin-B-deflcient Mouse Strain . . . . . . . . . . 23
2.2.1 Cloning of the Gene Targeting Vector . . . . . . . . . . 23
2.2.2 Gene Targeting in Embryonic Stem Cells . . . . . . . . 25
2.2.3 DNA Extraction from Cells in 96-Well Plates . . . . . 28
2.2.4 PCR Screening . . . . . . . . . . . . . . . . . . . . . . 28
2.2.5 Southern Blot . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.6 Blastocyst Injection . . . . . . . . . . . . . . . . . . . . 30
2.2.7 Embryo Transfer . . . . . . . . . . . . . . . . . . . . . 30
2.2.8 Animal Husbandry and Breeding . . . . . . . . . . . . 31
2.2.9 Genotyping . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3 Analysis of fetuin-B-deflcient Mice. . . . . . . . . . . . . . . . 32
i2.3.1 Western Blot . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.2 Immunohistochemistry . . . . . . . . . . . . . . . . . . 33
2.3.3 Collecting Mouse Oocytes . . . . . . . . . . . . . . . . 34
2.3.4 Primary Morphological Analysis . . . . . . . . . . . . . 35
2.3.5 Necropsy . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.3.6 Histology . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3.7 Weighing . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.3.8 Blood Pressure Measurement . . . . . . . . . . . . . . 37
2.3.9 Glucose Tolerance Test . . . . . . . . . . . . . . . . . . 37
2.3.10 Clinical Chemical Screening . . . . . . . . . . . . . . . 38
2.4 Recombinant Expression and Puriflcation . . . . . . . . . . . . 38
2.4.1 Cloning Murine Fetuin-B . . . . . . . . . . . . . . . . . 38
2.4.2 Generating a Fetuin-B Transfecting Adenovirus . . . . 41
2.4.3 Expression and Puriflcation of Fetuin-B. . . . . . . . . 41
2.5 In Vitro Analysis of Fetuin-B . . . . . . . . . . . . . . . . . . 42
2.5.1 Precipitation Inhibition Assay . . . . . . . . . . . . . . 42
2.5.2 Mink Lung Cell Reporter Assay . . . . . . . . . . . . . 43
2.6 Promoter Analysis . . . . . . . . . . . . . . . . . . . . . . . . 44
2.6.1 Cloning of Reporter Constructs . . . . . . . . . . . . . 44
2.6.2 Transfection of Reporter Constructs . . . . . . . . . . . 44
3 Results 47
3.1 Generating a Fetuin-B-deflcient Mouse Strain . . . . . . . . . 47
3.1.1 Gene Deletion and the Targeting Vector . . . . . . . . 47
3.1.2 Establishing the Screening Methods . . . . . . . . . . . 50
3.1.3 Gene Targeting in Embryonic Stem Cells . . . . . . . . 52
3.1.4 Screening for Recombinant ES Cell Clones . . . . . . . 54
3.1.5 Blastocyst Injection and Chimera . . . . . . . . . . . . 55
3.1.6 Embryo-Transfer and Breeding . . . . . . . . . . . . . 57
3.1.7 Conflrmation of Fetuin-B-deflcient Mice . . . . . . . . 58
3.2 Phenotyping Fetuin-B-deflcient Mice . . . . . . . . . . . . . . 59
3.2.1 Fertility . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.2.2 Anatomical and Morphological Analysis . . . . . . . . 67
3.2.3 Physiological Parameters . . . . . . . . . . . . . . . . . 74
3.2.4 Glucose Tolerance Test . . . . . . . . . . . . . . . . . . 74
3.2.5 Clinical Chemical Screening . . . . . . . . . . . . . . . 78
ii3.2.6 Summarizing the Phenotyping Results . . . . . . . . . 80
3.3 Recombinant Murine Fetuin-B . . . . . . . . . . . . . . . . . . 81
3.3.1 Cloning Recombinant Murine Fetuin-B . . . . . . . . . 81
3.3.2 Generating a Fetuin-B Transfecting Adenovirus . . . . 86
3.3.3 Expressing and Purifying Recombinant Fetuin-B . . . . 86
3.3.4 Precipitation Inhibition Assay . . . . . . . . . . . . . . 90
3.3.5 TGF-fl Reporter Activity in Mink Lung Cells . . . . . 90
3.3.6 Serum Concentration of Fetuin-B . . . . . . . . . . . . 93
3.3.7 Summarizing the Results of the in Vitro Data . . . . . 94
3.4 Promoter Analysis . . . . . . . . . . . . . . . . . . . . . . . . 95
3.4.1 Cloning and Analysis of the Fetub Promoter . . . . . . 95
4 Discussion 99
4.1 Generation of Fetuin-B-deflcient Mice . . . . . . . . . . . . . . 99
4.1.1 Gene Deletion . . . . . . . . . . . . . . . . . . . . . . . 99
4.1.2 Embryonic Stem Cells and Genetic Background . . . . 99
4.1.3 Screening Procedure . . . . . . . . . . . . . . . . . . . 100
4.1.4 Expression of Fetuin Family Proteins . . . . . . . . . . 101
4.1.5 Fetuin Serum Concentrations . . . . . . . . . . . . . . 101
4.2 Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.2.1 Vitality . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.2.2 Female Infertility . . . . . . . . . . . . . . . . . . . . . 102
4.2.3 Anatomy and Morphology . . . . . . . . . . . . . . . . 106
4.2.4 Physiology and Learning . . . . . . . . . . . . . . . . . 107
4.2.5 Clinical Chemistry Screening. . . . . . . . . . . . . . . 108
4.3 Recombinant Murine Fetuin-B . . . . . . . . . . . . . . . . . . 109
4.3.1 Eukaryotic Expression of Recombinant Fetuin-B . . . . 109
4.3.2 Fetuin-B Transfecting Adenovirus . . . . . . . . . . . . 110
4.3.3 Inhibition of Ectopic Calciflcation . . . . . . . . . . . . 110
4.3.4 TGF-fl Biology . . . . . . . . . . . . . . . . . . . . . . 111
4.4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Summary 113
Appendix 115
Pedigrees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Fetub Genomic Sequence . . . . . . . . . . . . . . . . . . . . . . . . 117
iiiAbbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
List of Figures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
References 142
Acknowledgements 143
Curriculum Vitae 145
ivChapter 1
Introduction
1.1 Fetuin Family of Proteins
For a long time the fetuin family of proteins consisted of a set of orthologous
serum proteins in humans, sheep, pigs, cows and rodents but also in birds
and reptiles. They were variably designated fetuin in sheep, pig and cow,
fi2-HS-glycoprotein (AHSG) in human, phosphoprotein of 63 kDa (pp63) in
rat or countertrypin in mouse and gerbil. However, their close sequence ho-
mology, especiallyasetof12cysteineresiduesatflxeddistancesandafetuin
motif (LETXCHXLDPTP), indicated that fetuin, AHSG, pp63 and coun-
tertrypin emerged from a single ancestral gene and resembled counterparts
(orthologous) among these species [1, 2, 3, 4, 5].
The fetuins were classifled as cystatin superfamily members because they
contain cystatin-like domains characterized by the distinct cysteine residues.
Further members of the cystatin superfamily, all sharing cystatin-like do-
mains, are the cystatins themselves, histidine-rich glycoproteins (HRG) and
kininogens (KNG). The cystatins are low-molecular-weight cysteine protease
inhibitors and prototypic for the cystatin superfamily [6, 7].
AstudythataimedatsystematiccloningofhepaticmRNAscorresponding
to in ammation-related genes in rat [8] revealed a second member of the
fetuinfamilybasedonsequencesimilarity. Sequenceanalysisshowedthatthe
novelproteincontainedbothfetuinsignatures,includingthesetof12cysteine
residues at flxed distances, perfectly conserved, and the fetuin motif that is
present in a truncated version (LETGCHVL) [9]. The novel fetuin, fetuin-
B was found in rat, mouse and humans, revealing both fetuin signatures
56 CHAPTER 1. INTRODUCTION
with a 61% overall sequence homology (strictly conserved residues) in these
species. Whencomparedtotheoriginalfetuin,fetuin-A,fetuin-Bshared17%
of strictly conserved amino acids and 24% of similar residues. The similarity
was signiflcant but much lower than the similarity between the rat, mouse
andhumanfetuin-A(58%strictlyconservedresidues). Thesequenceanalysis
indicated that fetuin-B was homologous, yet, clearly difierent from fetuin-A.
Therefore, the novel fetuins were considered a novel entity within the fetuin
family. The two paralogous were designated fetuin-A (comprising fetuin,
AHSG, pp63 and countertrypin) and fetuin-B (alias fetuin beta) [9].
1.1.1 Protein Structure
Three major domains (D1-D3) characterize fetuin proteins. Domain D1
and D2 are tandemly arranged cystatin-like domains containing 12 cysteine
residues at flxed distances, and are followed by a unique domain, D3, that is
only loosely conserved between species [10].
Infetuin-A,domainsD1andD2bothcontaindibasicmotifs, db1anddb2
respectively. They are thought to account for protease-inhibition domains
at least in human and bovine fetuin-A. Additionally, domain D1 contains a
calcium, TGF-fl and lectin binding sites [1, 11, 12]. Domain D3 can further
be subdivided into D3a, a hydrophobic, proline-rich N-terminal half, and
D3b, a C-terminal half including a connecting peptide, at least in humans
[1, 7, 13, 14].
The general organization of domains is maintained in fetuin-B, as shown
in Figure 1.1. However, the dibasic motifs db1 and db2 are only loosely
retained and the calcium-binding site in domain D1 is absent. Furthermore,
domain D3 does contain a proline-rich N-terminal but no connecting peptide
in the C-terminal half. However, fetuin-B contains a short homology to the
TGF-fl receptor type II (depicted in Figure 1.2) and two archetypal Kunitz-
type motifs, associated with protease-inhibitory activity, within domain D1
and D2 that is only present in fetuin-A domain D2 [9]. However, none of
these domains were functionally verifled.1.1. FETUIN FAMILY OF PROTEINS 78 CHAPTER 1. INTRODUCTION
Figure 1.1: Alignment of rat, mouse and human fetuin-B (f-B) with fetuin-
A (f-A) from various mammals (Olivier et al. [9]). White residues on black
background indicate identical or similar residues conserved in at least 10 out
of 11 sequences. Hyphens indicate gaps; boxes represent the proposed signal
peptides. The LETXCHXLDPTP fetuin signature is underlined. The 12 crit-
ical cysteine residues are marked with a dot. A CPG tripeptide conserved in
the cystatin superfamily is double-underlined. Disulphide bonds are indicated
byahorizontalconnectinglinebetweencysteineresidues;thebondconnecting
the flrst and ultimate cysteine is noted with a dashed line: The domains D1,
D2 and D3 (D3a and D3b) are marked with broken arrows. The connecting
peptide is over-lined and marked with CP. The potential calcium-binding site
is over-lined and marked with CBS. The dibasic peptides db1 and db2 are
over-lined.

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