Biochemical and genetic analysis of the adaptor protein SH3P7 [Elektronische Ressource] : insights from a newly generated knockout mouse / vorgelegt von Sabine Connert

Sabine Jourdain

Sujets

• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2003
• Nombre de lectures: 15
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

BIOCHEMICAL AND GENETIC ANALYSIS OF THE
ADAPTOR PROTEIN SH3P7:
INSIGHTS FROM A NEWLY GENERATED KNOCKOUT
MOUSE
Inaugural Dissertation
zur Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität Freiburg
im Breisgau
vorgelegt von
Sabine Connert
aus Mediasch in Siebenbürgen
Freiburg 2003Dekan der Fakultät Prof.Dr.Hans Kleinig
Promotionsvositzender Prof.Dr.Karl-Friedrich Fischbach
Betreuer der Arbeit Prof.Dr.Michael Reth
Leiter der Arbeit Prof.Dr.Jürgen Wienands
Korreferent PD Dr.Hermann Eibel
Dritter Prüfer Prof.Dr.Hans Ulrich Weltzien
Tag der Verkündigung des Prüfungsergebnisses: 21.Mai 2003
Die vorliegende Arbeit wurde von Oktober 1999 bis Januar 2002 am Max-Planck-
Institut für Immunbiologie in Freiburg und von Februar 2002 bis Januar 2003 an der
Universität-Bielefeld durchgeführt. Die Arbeit wurde unterstützt durch das
Ministerium für Wissenschaft, Forschung und Kunst des Landes Baden-Württemberg
(Forschungsschwerpunkt "Immundefizienzen") und durch die Deutsche
Forschungsgemeinschaft (SFB 549).Index
1 SUMMARY.........................................................................................................................................5
2 ZUSAMMENFASSUNG..................................................................................................................6
3 INTRODUCTION7
3.1 BCR RECEPTOR SIGNALING .......................................................................................................8
3.2 ENDOCYTOSIS OF THE BCR .....................................................................................................11
3.3 THE ACTIN-CYTOSKELETON AND ITS ROLE IN SIGNALING AND ENDOCYTOSIS.......................13
3.4 THE ADAPTOR PROTEIN SH3P7................................................................................................14
3.5 GENE TARGETING AND THE GENERATION OF KNOCKOUT MICE ..............................................17
4 METHODS.........21
4.1 RECOMBINANT DNA TECHNIQUES..........................................................................................21
4.1.1 Generation and transformation of competent bacteria................................................21
4.1.2 Production and purification of GST fusion proteins ....................................................22
4.1.3 Plasmid DNA amplification...........................................................................................23
4.1.4 Measurement of DNA concentrations...........................................................................24
4.1.5 Restriction digest and ligation of DNA.........................................................................24
4.1.6 Separation of DNA fragments on agarose gels ............................................................24
4.1.7 Purification of DNA from agarose gels ........................................................................24
4.1.8 Preparation of genomic DNA........................................................................................25
4.1.9 Amplification of DNA fragments from genomic DNA..................................................25
4.1.10 Genotyping of mice ........................................................................................................25
4.2 SOUTHERN BLOTTING AND HYBRIDIZATION............................................................................26
4.2.1 Southern blotting............................................................................................................26
324.2.2 Hybridization with P-labelled probes26
4.3 DNA SEQUENCING ...................................................................................................................27
4.4 CELL CULTURE OF MAMMALIAN CELLS ...................................................................................28
4.4.1 Cell lines.........................................................................................................................28
4.5 GENERATION OF KNOCKOUT MICE FROM ES CELLS................................................................29
4.5.1 General principle..29
4.5.2 Preparation of gelatin-coated tissue culture plates for ES cell culture ......................29
4.5.3 Preparation of mouse embryonic fibroblast and feeder layers for ES cell culture.....30
4.5.4 Cultivation of mouse ES cells........................................................................................31
4.5.5 Transfection of mouse ES cells......................................................................................31
4.5.6 Picking of ES cell clones ...............................................................................................32
4.5.7 Screening of ES cells for homologous recombinants ...................................................32
4.5.8 Freezing of ES cell clones in 96-well plates.................................................................32
4.5.9 Freezing of ES cell clones in single vials......................................................................33
4.5.10 Thawing ES cell clones from 96-well plates33
4.6 MOUSE STRAINS .......................................................................................................................33
4.7 FLOW CYTOMETRY...................................................................................................................33
4.7.1 Flow cytometric analysis33
4.7.2 Preparation of cell suspensions of different mouse organs .........................................34
4.7.3 Internalization assay......................................................................................................34
4.8 PROTEIN BIOCHEMISTRY ..........................................................................................................35
4.8.1 Stimulation of cells and preparation of cell lysates .....................................................35
4.8.2 Immuno-purification......................................................................................................35
4.8.3 SDS-PAGE and immunoblotting ...................................................................................35
4.9 HISTOCHEMISTRY.....................................................................................................................36
4.9.1 Preparation of cryo-sections.........................................................................................36
4.9.2 Staining of cryo-sections ...............................................................................................37
4.10 VARIOUS BUFFER COMPOSITIONS ............................................................................................37
4.11 DATABASE SOURCES AND ONLINE SEQUENCE SEARCH TOOLS................................................37
4.12 TEXT, IMAGE AND GRAPHICS PROCESSING SOFTWARE............................................................38
5 RESULTS.........................................................................................................................................39
5.1 SH3P7 BINDS TO HIP1R...........................................................................................................39
1Index
5.2 SH3P7 ASSOCIATES WITH SEVERAL COMPONENTS OF THE ENDOCYTIC MACHINERY ............41
5.3 B CELL ANTIGEN RECEPTOR STIMULATION REDUCES HIP1R-BINDING OF SH3P7 .................44
5.4 GENERATION OF A NEW MOUSE MODEL: CLONING AND CHARACTERIZATION OF SH3P7 GENE
FROM MICE...............................................................................................................................................49
5.5 GENERATION OF SH3P7 KNOCKOUT MICE................................................................................57
5.6 DELETION OF SH3P7 IN MICE IS NOT LETHAL DESPITE ITS WIDE EXPRESSION .......................64
-/-5.7 BCR INTERNALIZATION IS ACCELERATED IN SH3P7 MICE...................................................65
-/- +/-
5.8 MALE SH3P7 AND SH3P7 DEVELOP A FATAL DISEASE......................................................67
5.9 B CELL DEVELOPMENT OR MATURATION MIGHT BE IMPAIRED IN MALE SICKENED MICE ......74
5.10 PARALYSIS OF MALE SH3P7 KNOCKOUT MICE IS LIKELY CAUSED BY A NEUROLOGICAL THAN
A MUSCULAR DEFECT ..............................................................................................................................77
6 DISCUSSION....78
6.1 SH3P7/HIP1R INTERACTION IS THE FIRST STRUCTURAL LINK BETWEEN BCR STIMULATION,
THE ACTIN-CYTOSKELETON AND ENDOCYTOSIS.....................................................................................78
6.2 INTERACTION PARTNERS OF SH3P7: HOMOLOGIES FROM YEAST TO MAMMALS ...................82
6.3 THE SH3P7 KNOCKOUT MOUSE: A NEW ANIMAL MODEL TO STUDY SH3P7 FUNCTION.........89
6.4 SH3P7 IS IMPLICATED IN REGULATION OF BCR INTERNALIZATION ......................................90
6.5 ADULT MALE KNOCKOUT MICE AS WELL AS HETEROZYGOUS MICE DEVELOP A FATAL
DISEASE ..................................................................................................................................................92
6.6 PROTEINS WHICH MIGHT COMPENSATE FOR THE LACK OF SH3P7..........................................96
6.7 SIMILARITIES BETWEEN THE LOWE SYNDROME AND THE SH3P7 KNOCKOUT ........................98
6.8 OUTLOOK...99
7 REFERENCES.......................................................................................................................