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Conditional knockout of the L-type voltage gated calcium channel Cav1.3 via the FLEX switch [Elektronische Ressource] / presented by Katrin Bartels

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DISSERTATION submitted to the Combined Faculties for the Natural Scienc eMsa thaenmda tifocr s of the Ruperto-Carola University of Heidelbemarngy, Gerfor the degree of Doctor of Natural Sciences Presented by Diplom-Biologist Katrin Bartels born in Heidelberg, Germany Oral-examination: ______________ Conditional Knockout of the L-Type Voltage-gated Calcium Channel CaV1.3 via the FLEX Switch Referees: Prof. Dr. Hilmar Bading Prof. Dr. Dusan Bartsch Hiermit erkläre ich gemäß § 8 (3) b) muontido nscor)dn udnegr, Pdraoss ich die vorgelegte Dissertation selbst verfasst und mich dabeain dekereine ra ls der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe. I hereby declare according to § 8 (3t)he bd)o catnodra lc )d eogf ree regulations that I have written the submitted dissertation myself ainsd pirno ctehss I have used no other sources or materials than those expressly indicated. Mannheim, 23. Juli 2009 _ ___ ____ ____ ___ ____ ____ __ Katrin Bartels Acknowledgements This work was performed at the Central Insti tuMte ntaofl Health Mannheim (ZI) in the laboratory and under supervision of Prof. Dr. DBusaarntsch. Foremost, I would like to express my spec itaol Prtohf.a nkDsr. Dusan Bartsch for giving me the opportunity to work for my Ph.D.

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Published 01 January 2009
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DISSERTATION
submitted to the
Combined Faculties for the Natural Scienc eMsa thaenmda tifocr s
of the Ruperto-Carola University of Heidelbemarngy, Ger
for the degree of
Doctor of Natural Sciences












Presented by

Diplom-Biologist Katrin Bartels
born in Heidelberg, Germany



Oral-examination: ______________



Conditional Knockout of the L-Type Voltage-gated
Calcium Channel CaV1.3 via the FLEX Switch















Referees: Prof. Dr. Hilmar Bading
Prof. Dr. Dusan Bartsch





Hiermit erkläre ich gemäß § 8 (3) b) muontido nscor)dn udnegr, Pdraoss ich die vorgelegte
Dissertation selbst verfasst und mich dabeain dekereine ra ls der von mir ausdrücklich
bezeichneten Quellen und Hilfen bedient habe.

I hereby declare according to § 8 (3t)he bd)o catnodra lc )d eogf ree regulations that I have
written the submitted dissertation myself ainsd pirno ctehss I have used no other sources or
materials than those expressly indicated.


Mannheim, 23. Juli 2009 _ ___ ____ ____ ___ ____ ____ __
Katrin Bartels
Acknowledgements

This work was performed at the Central Insti tuMte ntaofl Health Mannheim (ZI) in the
laboratory and under supervision of Prof. Dr. DBusaarntsch.

Foremost, I would like to express my spec itaol Prtohf.a nkDsr. Dusan Bartsch for giving me
the opportunity to work for my Ph.D. thesis inl abhiorsa tory under excellent working
conditions and for providing me the interestjiengc tp, rofor his helpful suggestions and his
encouragement.

I am very grateful to Hilmar Bading for thsei ons uapned rvthie evaluation of my thesis.

Furthermore I would like to thank:
Kai Schönig for his scientific support regoraerdtincga lt hand technical aspects during the
thesis.
Tillmann Weber for his mentoring in the f,i rstth ey eianrtsroduction in the lab work and his
ongoing scientific support.
Vera Baier for her helpful remarks concemrmnuingof luthoere sicence techniques and for
her moral support.
Ariana Frömmig for generating the knockout anim als.
Thorsten Lau for his scientific support idni ffEeSr ecnteialtilon and microscopy.
Claus Beck and Uwe Leimer for the introducS ticone linl Eculturing.
Celine Lentrodt for her help during the thcel otnarigneg tionf g construct.
All former and recent lab members for tche suscppioertn,t ifciontinuous help and for the
nice time in the lab.
All animal care takers at the ZI, espSeiceigaell,y taPkeintegr care of the transgenic mice.
All members of the graduate college 7u9p1p orftor itnh etier cshnical or scientific questions.
The graduate college 791 for the financ ial support.

Summary
Summary
L-type voltage-gated calcium channels (VGaC Ccs)e nptrlalay role in regulating intracellular
2+calcium ()C a concentrations, thereby contributing to ansidguncalti on trin many
electrically excitable cells. TheV 1.L3-t yipse ecxphraennseseld Cian neurons, in hair cells of
the inner ear, in heart tissue and in pcaneclrelast.i cM icß-e with a ubiquitous deletion of the
-/-pore-forming α1-subunit (VC1a.3) showed that VC1a.3 has cardiac pace maker activity and
2+controls the neurotransmitter release in tah.e Fucrtohcehrlmoere, Ca influx through this
channel was proposed to be involved in atnexdi etbye-rheavliaour and in consolidation of
contextually conditioned fear. Aim of my worko wgaasi n tmore insight into the
physiological role Vo1f .3C ain different cell types and tissu eis,n enespuerocns.i alTlo y
circumvent the phenotypes of the conventionaoult kmniocek like deafness and heart
insufficiency which may interfere with baenahlavyisoiusr,a l we decided to generate a
conditional CV1a.3α1 knockout mouse using Cthre/ loxP-based FLEX switch system. By a
Cre-mediated inversion of the targeted locus,at egthyi s cstourpled the ablation of the
CaV1.3α1 gene to the expression of the reporGteFPr, gtehenere bey mirroring the exact
flexexpression pattern of the endogenouVs1 .3Ca gene in all tissues. HeterVo1z.y3g-ouGFsP C a
mice were bred with “Cre-deleter” mice Cirne wrehicomhb itnhaese is ubiquitously
expressed. We could show that,C rea fterx pression, the FLEX switch occurred ine fficiently
vitro and in vivo and that the reporter gene was driven by etnhoeus eCnaVd1og.3α1
promoter. Immunohistochemical analysis revealePd eexGpFression in heart tissue as well as
in many brain areas like the olfactory breulbebl, ltuhem, csueperior colliculus, brain regions
associated with emotional behaviour and sensorcy espsriong. In contrast to previously
reported CVa1.3 expression, only moderate eGFP staining nwd as inf ouhippocampal
regions. An unexpected observation was the eiGnFtPe nseex pression in the peri- and
subventricular zone, suggesting a Vr1ol.3eα 1of iCna adult neurogenesis and neural stem cell
flexproliferation. In parallel, we cV1r.o3ss-eGFdP C amice to CaMKIIa-mice, thereby obtaining
specific eGFP expression in the forebrain taoc ctheord inegxpression of the CaMKIIa
promoter.
This mouse model offers a great potential rto ifnuvrethsetigate the distribution and function
2+of the L-type C achannel V1C.3a in various tissues.


Zusammenfassung
Zusammenfassung
L-typ spannungsgesteuerte Calciumkanäle (VGCCl)e ns pieeine zentrale Rolle in der Regulierung
2+der intrazellulären KConazentration und tragen somit zur Signalveruanrgb eitn vielen
elektrisch erregbaren Ze lDleer n L-btyepi .Kanal V1C.a3 wird in Neuronen, in Haarzellen des
Innenohrs, im Herzgewebe und in ß-Zellen asd ees xpPrainmkrieert. Mäuse mit einer ubiquitären
-/-Deletion der porenbildenden α1-UntereinhVe1i.3t ) (Czaeigten, dass deVr1 .3C a Kanal
Herzschrittmacher-Funktionen besitzt und die raNnesumirotter-Ausschüttung in der Cochlea
kontrollier t.Des Weiteren wird dieser Kanal mit dem Äntgsvsetlrhiaclhtkeni und
Konsolidierung von kontextabhängig konditioniertAenrg st in Verbindung gebracht. Ziel dieser
Arbeit war es, weitere Einblicke in dciehe pRhoylsiloleo gvisoVn1 .3C a in verschieden Zelltypen
und Geweben, im Besonderen in Neuronen, zu. eDrah alkontevenntionelle Knockout-Mäuse
Phänotypen wie Taubheit und Herzinsuffizienz , zediege nVerhaltensanalysen beeinträchtigen
können, haben wir uns entschlossen, diese nz u unudm geehiene konditionaleV 1.C3aα1
Knockout-Maus mithilfe deCsr e/loxP-basierten „FLEX switch“ Systems zu genericerhe ne. inD ur
Cre-vermitteltes Invertieren des „gefloxten“p pAellt edlies seko Strategie die Inaktivierung des
CaV1.3α1 Gens an die Expression des Reportergen s weodGuFrPc,h das exakte
Expressionsmuster des endogenen V1C.a3α1 Gens in allen Geweben gespiegelt wird.
flexHeterozygote CVa1.3-GFP Mäuse wurden mit “Cre-Deleter”-Mäusen verlpcaarhte, Cwdire-
Rekombinase ubiquitär exprimie rWeinr. konnten zeigen, dass nCacre-hE xpression der FLEX
„switch“i n vitro und in vivo effizient erfolgte und dass das Reportergemn evnodno gedenen
CaV1.3α1 Promotor getrieben wurde. Immunhistochemi scAnhaelysen ließen eGFP-Expression
im Herzgewebe und vielen Hirnarealen erkdeenmn enol, fakwtoiries chen Bulbus, Cerebellum,
Superior Colliculus und Regionen, die mit em emoVteiornhalten und sensorischer
Verarbeitung assoziiert sind. Im Gegensatz zue kadnentre nb CaV1.3 Expression, zeigte sich in
den hippokampalen Hirnregionen nur eine gemäßGiFgPt-eFä rbeung. Eine unerwartete
Beobachtung war die intensive eGFP-Expressri onp eirni -d eund subventrikulären Zone, was auf
eine Funktion von VC1a.3 in adulter Neurogenese und neuronaler pSrtoalmimfzerlatlion
flexhinweisen könnte. Parallel verpaarteVn1 .3w-iGrF PCa Mäuse mit CaMKIIa-Mäusen, wobei wir
eine spezifische eGFP-Expression im Vorderhirbne rieni nsÜtimmung mit der Expression des
CamKIIa-Promoters ausmachen konnten.
flexDas CaV1.3-GFP Mausmodel stellt somit ein großes Potentialie dFaru,n ktidon and
Verbreitung des L-typ KalziumkanalVs1 .3C aweiter aufzuklären.
Contents
Contents
1 Introduction........................................................................................................................1
1.1 Calcium as Second Messenger.............................................................................................. 1
2+ 1.1.1 Ca Signalling................................................................................................................................... 3
2+1.1.2 Ca Signalling in Neuronal Cells ..................................................................................................... 4
1.2 The L-type Voltage-gated Calcium Channel Ca 1.3.......................................................... 6 V
1.2.1 Calcium Channel Diversity ............................................................................................................... 6
1.2.2 Structural Organization of L-type Ca 1.3 Channels ......................................................................... 8 V
1.2.3 L-type Ca 1.3 Channel Properties................................................................................................... 10 V
1.3 Conditional Knockout of the Ca 1.3 α1 Subunit Gene.................................................... 16 V
1.3.1 The Cre-loxP Recombination System............................................................................................. 17
1.3.2 Conditional Gene Inactivation via the FLEX Switch...................................................................... 19
1.4 Aim of the Thesis ................................................................................................................. 22
2 Material............................................................................................................................23
2.1 Laboratory Equipment ....................................................................................................... 23
2.1.1 Microscopy...................................................................................................................................... 23
2.1.2 Agarose Gelelectrophoresis............................................................................................................. 24
2.1.3 Cell Culture ..................................................................................................................................... 24
2.2 Consumables ........................................................................................................................ 24
2.3 Chemicals ............................................................................................................................. 24
2.4 Antibiotics ............................................................................................................................ 25
2.5 Enzymes and Recommended Buffers ................................................................................ 25
2.6 Reaction buffers for restriction endonucleases................................................................. 26
2.6.1 Fermentas Buffers ........................................................................................................................... 26
2.6.2 New England Biolabs Buffers......................................................................................................... 27
2.7 Antibody Staining................................................................................................................ 27
2.7.1 Antibodies ....................................................................................................................................... 27
2.7.2 Compounds for Antibody Staining.................................................................................................. 28
2.8 Nucleic Acids........................................................................................................................ 28
2.8.1 Nucleotids ....................................................................................................................................... 28
2.8.2 DNA Markers.................................................................................................................................. 28
2.8.3 Primers ............................................................................................................................................ 28
2.8.4 Plasmids .......................................................................................................................................... 29
2.9 Bacteria Strains ................................................................................................................... 29
2.10 Cell Lines.............................................................................................................................. 30
2.11 Mouse Lines ......................................................................................................................... 30
2.12 Buffers, Media and Solutions ............................................................................................. 31
2.12.1 Cell Culture Media...................................................................................................................... 33
3 Methods............................................................................................................................34
3.1 Microbiological Methods .................................................................................................... 34
3.1.1 Transformation of Bacteria ............................................................................................................. 34
3.2 Isolation and Purification of DNA ..................................................................................... 35
3.2.1 Isolation of Plasmid DNA (Miniprep)............................................................................................. 35
3.2.2 Isolation of Plasmid DNA on a large scale (Maxiprep) .................................................................. 36
3.2.3 Isolation of genomic DNA from tail tissue of the mouse................................................................ 37
3.2.4 Phenol-Chloroform extraction of DNA........................................................................................... 38
3.2.5 Photometric analysis of concentration and purity of nucleic acids ................................................. 38
3.2.6 DNA Extraction from Agarose Gel................................................................................................. 39
3.2.7 Extraction of DNA by Agarose Digestion....................................................................................... 40
3.3 Purification of DNA............................................................................................................. 41
3.4 Analysis of DNA................................................................................................................... 42
3.4.1 Fragmentation of DNA with Restriction Enzymes.......................................................................... 42
3.4.2 Agarose Gel Electrophoresis ........................................................................................................... 42
3.4.3 Staining of Nucleic Acid Agarose Gels with Ethidium Bromide.................................................... 45
3.4.4 Oligonucleotide Annealing.............................................................................................................. 45
3.4.5 Dephosphorylation of 5’-Ends from DNA by Alkaline Phosphatase.............................................. 45
3.4.6 Blunting of DNA Fragments with 5’- or 3’ – Protruding Ends....................................................... 46
3.4.7 Ligation of DNA Fragments............................................................................................................ 46
3.4.8 Polymerase Chain Reaction............................................................................................................. 47
3.4.9 Colony PCR .................................................................................................................................... 48
3.4.10 Sequencing of DNA.................................................................................................................... 48
3.5 In vitro Modification and Recombination in Bacteria..................................................... 49
3.5.1 Transformation of BAC DNA into Recombinant Strains................................................................ 50
3.5.2 Recombination through Temperature Control................................................................................. 50
3.5.3 Excision through Arabinose Induction............................................................................................ 51
3.6 Immunohistochemistry ....................................................................................................... 51
3.6.1 DAB Method................................................................................................................................... 51
3.6.2 Immune Fluorescence ..................................................................................................................... 52
3.7 Cultivation of Mammalian Cells ........................................................................................ 52
3.7.1 Hela Cells ........................................................................................................................................ 53
3.8 DNA Transfer in Mammalian Cells................................................................................... 53
3.8.1 Transient Transfection by Lipofection ............................................................................................ 53
3.9 Generation of Genetically Modified Mice ......................................................................... 53
3.9.1 Preparation of Mouse Embryonic Fibroblasts (MEFs).................................................................... 53
3.9.2 Cultivation of Embryonic Stem Cells (ES cells) ............................................................................. 54
3.9.3 Homologous recombination in ES cells .......................................................................................... 55
3.9.4 Identification of Genetically Modified ES cells .............................................................................. 58
3.9.5 Cre-mediated Recombination in ES Cells....................................................................................... 59
3.9.6 Preparation of positive ES cells for blastocyst injection ................................................................. 60
3.9.7 Germ line transmission of chimeric mice........................................................................................ 61
3.9.8 Animal husbandry and breeding...................................................................................................... 61
4 Results...............................................................................................................................63
flex
4.1 Generation of the Ca 1.3-GFP Mouse Line.................................................................. 63 V
flex4.1.1 Construction of the Targeting Vector BAC.Ca 1.3-GFP ............................................................ 63 V
flex
4.1.2 Functionality of the Targeting Construct BAC.Ca 1.3-GFP in vitro.......................................... 69 V
flex
4.1.3 Production of Ca 1.3-GFP -Targeted of ES Cell Clones ............................................................ 70 V
flex4.1.4 Generation of the Mouse Line Ca 1.3-GFP ................................................................................ 72 V
flox4.1.5 Genotyping of Ca 1.3-GFP mice................................................................................................ 74 V
4.1.6 Removal of the Neomycin cassette by Flpe-recombination............................................................ 74
flex
4.2 Conditional Knockout of the Ca 1.3α1 subunit via the FLEX Switch in Ca 1.3-GFP V V
mice 76
flex
4.2.1 PCR Analysis of Ca 1.3-GFP x Cre-deleter mice....................................................................... 76 V
4.2.2 Analysis of the mRNA Expression by RT-PCR.............................................................................. 77
switch4.2.3 Expression of eGFP Protein in Ca 1.3-GFP Mice ................................................................... 79 V
flex
4.2.4 eGFP Expression in Ca 1.3-GFP x CaMKIIα-Cre mice............................................................. 83 V
4.2.5 Analysis of eGFP Protein Expression by Double Immunofluorescence ......................................... 85
5 Discussion.........................................................................................................................92
flex
5.1 Targeting Strategy of the Ca 1.3-GFP Construct ........................................................ 92 V
flex
5.2 ES Cell Targeting of Ca 1.3-GFP .................................................................................. 95 V
5.3 Conditional Knockout of Ca 1.3α1 by the Cre-mediated FLEX switch ........................ 96 V
5.4 mRNA-Expression of Ca 1.3.............................................................................................. 98 V
5.5 Expression pattern of Ca 1.3α1 mRNA represented by eGFP staining......................... 99 V
5.5.1 eGFP Expression in Neurons ........................................................................................................ 100
5.5.2 eGFP Staining in the Periventricular zone .................................................................................... 103
5.5.3 eGFP Expression in Other Tissues................................................................................................ 104
5.6 Conclusion and Outlook ................................................................................................... 105
6 Abbreviations..................................................................................................................107
7 References......................................................................................................................110