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Gene$C Knockout of Foxg1 in Murine Pluripotent Stem Cells and Evalua$On of Its Func$Onality Via Neuronal Differen$A$On in Vitro

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Gene$C Knockout of Foxg1 in Murine Pluripotent Stem Cells and Evalua$On of Its Func$Onality Via Neuronal Differen$A$On in Vitro Gene$c knockout of Foxg1 in murine pluripotent stem cells and evalua$on of its func$onality via neuronal differen$a$on in vitro A thesis submi:ed for the degree of Doctor of Natural Sciences (Dr. rer. nat.) in the subject of Molecular Biology by Eva Maria Mall, M.Sc. Animal Biology April 2017 Interna$onal PhD program ‘Regenera$ve Sciences’ Department of Biotechnology, Friedrich-Loeffler-Ins@tute for Farm Animal Gene@cs Mariensee, Neustadt am Rübenberge The PhD project was funded by the DFG trough the Cluster of Excellence REBIRTH (CRU. 9.1) Acknowledged by the PhD commiJee and head of Hannover Medical School President: Prof. Dr. Christopher Baum Supervisor: Prof. Dr. Heiner Niemann, Ins$tute for Farm Animal Gene$cs, Friedrich-Loeffler-Ins$tute Mariense, Neustadt am Rübenberge 1st Co-Supervisor: Prof. Dr. Achim Gossler, Ins$tute for Molecular Biology, Hannover Medical School, Hannover 2nd Co-Supervisor: Prof. Dr. Michael O:, Department of Gastroenterology, Hepatology & Endocrinology, Hannover Medical School Hannover External expert: Prof. Dr. Ulrich Rüther, Ins$tute for Animal Developmental and Molecular Biology, Heinrich-Heine University, Düsseldorf Internal expert: Prof. Dr. Thomas Moritz, Ins$tute of Experimental Hematology, Hannover Medical School, Hannover Day of public defence: 23rd June 2017 Table of Content 1 Abstract .............................................................................................................1 2 Introduc$on.......................................................................................................2 2.1 Project context.........................................................................................................2 2.2 Pluripotent stem cells and gene$c modifica$ons ....................................................5 2.2.1 Pluripotent stem cells ..............................................................................................5 2.2.2 CRISPR/Cas mediated gene knockout ......................................................................9 2.3 Neuronal differen$a$on ........................................................................................13 2.3.1 Important aspects of cor$cogenesis in vivo...........................................................13 2.3.2 Cor$cogenesis of pluripotent stem cells - Default model ......................................15 2.4 Forkhead box transcrip$on factor G1 (Foxg1)........................................................19 2.4.1 Structure and func$ons of Foxg1 in mice ..............................................................19 2.4.2 Foxg1 knockout in mice .........................................................................................21 2.4.3 FOXG1 related diseases in humans ........................................................................22 2.5 Aim of this study ...................................................................................................24 3 Materials and methods ....................................................................................25 3.1 Project workflow....................................................................................................25 3.2 Cell culture .............................................................................................................26 3.2.1 Mouse pluripotent stem cell lines (mPSCs)............................................................26 3.2.2 Mouse pluripotent stem cell culture......................................................................27 3.2.3 HEK293T cell culture and transfec$on with FOXG1 expression plasmid................29 3.3 CRISPR/Cas9 mediated knockout of Foxg1 ............................................................30 3.3.1 CRISPR/Cas9 plasmid genera$on ...........................................................................30 3.3.2 Genera$on of Foxg1 knockout mPSCs ...................................................................32 3.3.3 Analysis of knockout phenotype ............................................................................34 3.4 Neuronal differen$a$on towards telencephalic progenitors.................................36 3.4.1 Differen$a$on of mPSCs towards telencephalic progenitors.................................36 3.4.1.1 Preliminary differen$a$on protocol.......................................................................38 3.4.1.2 Final differen$a$on protocol .................................................................................39 3.4.2 Analysis of embryoid body like structure size ........................................................40 3.4.3 Analysis of Foxg1 expression using Venus fluorescence in reporter mESC ............40 3.4.4 Analysis of protein expression ...............................................................................41 3.4.4.1 Different protein isola$on methods.......................................................................41 3.4.4.2 SDS PAGE and Western blot analysis......................................................................42 3.4.4.3 Mass spectrometry analysis...................................................................................44 3.4.4.4 Immunoprecipita$on .............................................................................................45 3.4.5 Analysis of neuronal gene expression....................................................................46 3.4.5.1 Isola$on of RNA, DNA, Protein ..............................................................................47 3.4.5.2 Reverse transcrip$on reac$on ..............................................................................48 3.4.5.3 End-point reverse transcrip$on (RT) PCR ..............................................................48 3.4.5.4 Quan$ta$ve real-$me (q)PCR................................................................................49 3.5 Sta$s$cs .................................................................................................................51 4 Results .............................................................................................................52 4.1 CRISPR/Cas9 mediated knockout of Foxg1 in mouse pluripotent stem cells .........52 4.2 Preliminary tests of neuronal differen$a$on towards cor$cal progenitors...........55 4.2.1 Establishment of a neuronal differen$a$on protocol ............................................55 4.2.2 Op$miza$on of embryoid body like structure forma$on ......................................59 4.2.3 Valida$on of differen$a$on protocol towards cor$cal progenitors.......................63 4.2.3.1 Growth of embryoid body like structures over $me..............................................63 4.2.3.2 Analysis of Venus fluorescence ..............................................................................64 4.2.3.3 Analysis of neuronal gene expression by qPCR......................................................66 4.2.4 Foxg1 protein expression analysis..........................................................................68 4.2.4.1 Foxg1 C-Terminal an$body (ab18259) ...................................................................70 4.2.4.2 Foxg1 N-Terminal an$body (ab86292)...................................................................73 4.2.4.3 Foxg1 Central an$body (polyclonal, SAB1307246) ................................................74 4.2.4.4 Foxg1 Central an$body (monoclonal, MABD79)....................................................75 4.3 Analysis of neuronal differen$a$on in Foxg1 wildtype and knockout mPSCs........76 4.3.1 Pluripotent stem cell lines included in the analysis ...............................................76 4.3.2 Analysis of growth kine$cs in Foxg1 wildtype and knockout mPSCs .....................78 4.3.3 Analysis of size of the derived embryoid body like structures ...............................79 4.3.4 Analysis of neuronal gene expression by qPCR......................................................81 4.3.4.1 Differen$a$on efficiencies in different Foxg1 wildtype mPSCs..............................81 4.3.4.2 Comparison of Foxg1 knockout mPSCs to their wildtype controls.........................84 5 Discussion........................................................................................................86 5.1 Methodological aspects .........................................................................................87 5.2 Analysis of neuronal differen$a$on in various pluripotent stem cell lines............92 6 Conclusions and outlook ..................................................................................98 7 References .......................................................................................................99 8 Appendix .......................................................................................................114 8.1 Supplementary materials & protocols .................................................................114 8.1.1 Cell lysis buffer for DNA prepara$on....................................................................114 8.1.2 Heat-shock transforma$on of competent bacteria..............................................114 8.1.3 Protein isola$on and western blot analysis .........................................................114 8.2 Supplementary results .........................................................................................116 8.2.1 Growth kine$cs ....................................................................................................116 8.2.2 Size of derived embryoid bodies..........................................................................116
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