"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-de¯cient 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-de¯cient Mice . 32 i 2.3.1 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 Puri¯cation . 38 2.4.1 Cloning Murine Fetuin-B . 38 2.4.2 Generating a Fetuin-B Transfecting Adenovirus . 41 2.4.3 Expression and Puri¯cation 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-de¯cient 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 Con¯rmation of Fetuin-B-de¯cient Mice . 58 3.2 Phenotyping Fetuin-B-de¯cient 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 ii 3.2.6 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-¯ 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-de¯cient 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 Calci¯cation . 110 4.3.4 TGF-¯ Biology . 111 4.4 Outlook . 112 Summary 113 Appendix 115 Pedigrees . 115 Fetub Genomic Sequence . 117 iii Abbreviations . 125 List of Figures . 128 List of Tables . 129 References 142 Acknowledgements 143 Curriculum Vitae 145 iv Chapter 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, ®2-HS-glycoprotein (AHSG) in human, phosphoprotein of 63 kDa (pp63) in rat or countertrypin in mouse and gerbil. However, their close sequence ho- mology, especially a set of 12 cysteine residues at ¯xed distances and a fetuin 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 classi¯ed 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]. A study that aimed at systematic cloning of hepatic mRNAs corresponding to inflammation-related genes in rat [8] revealed a second member of the fetuin family based on sequence similarity. Sequence analysis showed that the novel protein contained both fetuin signatures, including the set of 12 cysteine residues at ¯xed 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 5 6 CHAPTER 1. INTRODUCTION with a 61% overall sequence homology (strictly conserved residues) in these species. When compared to the original fetuin, fetuin-A, fetuin-B shared 17% of strictly conserved amino acids and 24% of similar residues. The similarity was signi¯cant but much lower than the similarity between the rat, mouse and human fetuin-A (58% strictly conserved residues). The sequence analysis indicated that fetuin-B was homologous, yet, clearly di®erent 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 ¯xed distances, and are followed by a unique domain, D3, that is only loosely conserved between species [10]. In fetuin-A, domains D1 and D2 both contain dibasic motifs, db1 and db2 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-¯ 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-¯ 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 veri¯ed. 1.1. FETUIN FAMILY OF PROTEINS 7 8 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 by a horizontal connecting line between cysteine residues; the bond connecting the ¯rst 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. 1.1. FETUIN FAMILY OF PROTEINS 9 Figure 1.2: Modeled binding of human TGF-beta to its receptor type II (structure software Pymol 0.9V). The TGF-¯ binding site is depicted in green. The homology between the receptor and fetuin-B is depicted in yellow. Note that it is not part of the TGF-¯ binding site.