Homologous recombination and directed differentiation in medaka ES cells development of vector systems

HOMOLOGOUS RECOMBINATION AND DIRECTED DIFFERENTIATION IN MEDAKA ES CELLS: DEVELOPMENT OF VECTOR SYSTEMS LU WENQING (B.Sc SHANGHAI JIAO TONG UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements Herewith I express my utmost gratitude to my supervisor Assoc Prof Hong Yunhan for his advice, guidance, inspiration and patience during the period I have been working with him I thank Professor Georg Kröhne for his plasmid pEGFP-C2-lap2-503-657 which was used in constructing the bicistronic vector pCVmpf, and Mr WJ Wang for his data and plasmids on Rad51 and Dmc1 I thank Madam Veronica Wong, our laboratory officer for her kind assistance in all administrative matters I would like to thank Madam Deng Jiaorong for her assistance in all matters related to the aquarium and fish management I extend my thank to all my laboratory mates in the Developmental Genetics Laboratory, Dr Zhao Haobin, Dr Liu Tongming, Tiansheng, Menghuat, Hongyan, Dr Qin Lianju, Jane, Mingyou, Zhengdong, Liu Rong, Leon and Dr Zeng Zhiqiang,for their help during the course of my project Their presence has created an enjoyable environment Lu Wenqing July 20045 i TABLE OF CONTENTS ACKNOWLEDGEMENTS SUMMARY LIST OF ABBREVIATIONS I VI VIII LIST OF FIGURES X LIST OF TABLES XI CHAPTER INTRODUCTION 1.1 DOUBLE STRAND BREAKS AND DNA REPAIR 1.2 MODELS OF DSB REPAIR 1.3 PATHWAYS AND GENES INVOLVED IN DSB REPAIR 1.4 MEDAKA AS A MODEL ORGANISM 11 1.5 EMBRYONIC STEM CELLS IN MEDAKA 16 1.6 MELANOCYTES AND MITF 17 1.7 DIRECTED DIFFERENTIATION OF MELANOCYTES FROM MEDAKA ES CELL 19 1.8 GOALS 20 CHAPTER 2: MATERIALS AND METHODS 21 2.1 21 MATERIALS 2.1.1 Experimental animals 21 2.1.2 Cell lines 21 2.1.3 Bacteria 22 2.1.4 Enzymes 22 2.1.5 Antibodies 22 2.1.6 Oligo nucleotides 23 ii 2.1.7 24 METHODS 24 2.2 Plasmid 2.2.1 Molecular Cloning 24 2.2.1.1 RNA Isolation 24 2.2.1.2 cDNA synthesis 25 2.2.1.3 Polymerase Chain Reaction 26 2.2.1.4 Agarose gel electrophoresis 27 2.2.1.5 Recovery of DNA fragments from agarose gel 27 2.2.1.6 Ligation of DNA fragment into PGEM-vector 28 2.2.1.7 E.coli transformation 29 2.2.1.8 Plasmid DNA isolation and test digestion 30 2.2.1.9 Digestion of DNA with restriction endonuclease 31 2.2.1.10 DNA sequencing and analyses 31 2.2.2 Plasmid construction 33 2.2.2.1 Construction of pCV-DMC1-N-His 33 2.2.2.2 Construction of pCV-Rad51-N-His 33 2.2.2.3 Construction of pSTK-zlap2egfp 34 2.2.2.4 Construction of pHR/EJ 3.2 34 2.2.2.5 Construction of pN3 35 2.2.2.6 Construction of pN4 35 2.2.2.7 Construction of pCVmpf 36 2.3 PROTEIN BIOLOGY 37 2.3.1 Protein isolation from tissues and cell lines 37 2.3.2 Protein assay 38 2.3.3 SDS-Polyacrylamide gel electrophoresis 38 iii 2.3.4 2.4 Western blotting CELL BIOLOGY 39 41 2.4.1 Buffers and media 41 2.4.2 Preparation of embryo extract and fish serum 42 2.4.3 Thawing, freezing and maintenance of cell lines 42 2.4.4 Gene transfer into medaka cell lines and transient expression 43 2.4.5 Drug selection of transfected cells 43 2.4.6 Determination of HR and NHEJ activity 44 CHAPTER RESULTS 3.1 45 STRATEGY AND EXPERIMENTAL DESIGN OF THE SINGLE PLASMID GENE ACTIVITY ASSAY SYSTEM 45 3.2 CHARACTERIZATION OF PHR/EJ 3.2 47 3.2.1 Transient transfection into cell lines 47 3.2.2 Homologous recombination product 48 3.3 OVEREXPRESSION OF RAD51 49 3.3.1 Rad51 was overexpressed at mRNA level 49 3.3.2 Western blotting 50 3.4 OVEREXPRESSION OF DMC1 50 3.4.1 Dmc1 was overexpressed at mRNA level 50 3.4.2 Western blotting 53 3.5 CELLULAR ACTIVITY ASSAY OF RAD51 AND DMC1 ON HR IN THREE CELL LINES 54 3.6 EXPERIMENT APPROACH TO ENRICH DIFFERENTIATING MELANOCYTE LINEAGE 3.7 61 BICISTRONIC PLASMID CAN DIRECT AND ENRICH DIFFERENTIATION OF iv MELANOCYTES FROM MES1 61 CHAPTER DISCUSSION 63 4.1 SINGLE PLASMID SYSTEM FOR ASSAY OF CELLULAR ACTIVITY IN HR AND NHEJ 63 4.2 EFFECTS OF RAD51 AND DMC1 ON DSB REPAIR 64 4.3 DIRECTED DIFFERENTIATION OF MELANOCYTES FROM MES1 70 4.4 IMPLICATIONS AND FUTURE WORK 71 CONCLUSION 73 REFERENCE LIST 75 APPENDICES 86 PLASMID MAP 86 v Summary DNA double strand breaks (DSB) repair operates in homology-dependent and –independent ways Major pathways include synthesis-dependent strand annealing (SDSA), double strand break repair (DSBR), single strand annealing (SSA) and non homologous end joining (NHEJ) Different genes are involved in each pathway and the output products are also different To establish a single plasmid system for rapid assay of cellular activities for homologous recombination (HR) and NHEJ, a plasmid, pHR/EJ 3.2, was constructed It contains two partial repeats of red fluorescent protein (RFP) The two partial repeats are separated by a cassette expressing green fluorescent protein (GFP) After HR, linearized plasmid will generate RFP in the cytoplasm Plasmid products by NHEJ will give rise to GFP in the nucleus His-tagged fusion protein Rad51 and Dmc1 were overexpressed in three medaka cell lines Overexpressions were confirmed on mRNA level and protein level Effects of Rad51 and Dmc1 were studied by overexpression in three cell lines followed by fluorescent cell counting and statistical analysis Overexpressed Rad51 and Dmc1 proteins have similar effects on DSB repair But differences between different cell lines were observed Embryonic stem cell (ES) line is a unique cell line that can divide infinitely and differentiate into virtually all types of cells Mechanism of differentiation is still largely unknown Melanocytes are among the best studied cells in terms of lineage differentiation Mitf, a gene that is necessary to direct ES cells into melanocytes, provides a good opportunity to study differentiation But the co-transfection vi system was inefficient to enrich differentiating cells A bicistronic plasmid was constructed that can direct ES cells to differentiate and confer drug resistance and GFP expression for screening The plasmid was highly efficient and specific to enrich the differentiating transgenic cells vii List of abbreviations ATP Adenosine Triphosphate cDNA Complementary DNA DSB Double Strand Break DSBR Double Strand Break Repair dsDNA Double Stranded DNA EB Ethidium Bromide EDTA Ethylenediamine-Tetraacetic Acid ES Embryonic Stem GFP Green Fluorescent Protein HDR Homologous Dependent Repair HR Homologous Recombination LB Luria Bertani NHEJ Non Homologous End Joining ORF Open Reading Frame pac Puromycin Acetyltransferase PAGE Polyacrylamide Gel Electrophoresis PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction RFP Red Fluorescent Protein RT-PCR Reverse Transcription-Polymerase Chain Reaction SDSA Synthesis-Dependent Strand Annealing viii ssDNA Single Stranded DNA SSA Single Strand Annealing TAE Tris-Acetate-Edta TMEMD N,N,N’,N’-Tetramethylethylenediamine x-gal 5-Bromo-4-Chloro-3-Indoyl-Β-D-Galactoside ix microphthalmia-associated transcription factor (mitf) Drug selection of medaka embryonic stem cells transiently transfected with this 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of pHR/EJ 3.2 Negative control of pHR/EJ 3.2 Bicistronic vector for generation and enrichment of transgenic differentiating melanocytes C MV H is tag pC V-D MC 1-N-His 5175 bp D MC1 K an /N eo SV40 p olyA 86 C MV H is tag pC V-Rad51-N-His R ad 51 5175 bp K an /N eo SV40 p olyA SVTK pU C ori EGF P H SV TK p olyA zlap 2-503-657 pSTK-zlap2egfp 6248 bp K ana/N eo SV40 p olyA SV 40 early p romo ter SV40 p olyA 3' mR N A en d C MV Pac K an/N eo partial D sRed SV40 p olyA pHR/EJ-3.2 10527 bp SV40 p olyA SV40 p olyA D sR ed partial pac zlap svtk EGF P 87 C MV Pac pN3 5200 bp p artial D sR ed K an /N eo SV40 p olyA C MV SV40 K an/N eo SV40 p olyA pN4 9113 bp SV40 p olyA zlap D sR ed EGF P partial pac svtk C MV promo ter mitf-m SV40 pCVmpf 7760 bp C MV promo ter K ana/n eo pf2 SV40 po lyA SV40 po lyA 88 ... RELATIVE ACTIVITY OF HOMOLOGOUS RECOMBINATION VERSUS END JOINING IN SG3 CELL LINE 62 TABLE RELATIVE ACTIVITY OF HOMOLOGOUS RECOMBINATION VERSUS END JOINING IN SOK CELL LINE 63 xi Chapter Introduction... fish Yes Yes No Chimeric fish Yes Yes No Gynogenesis Yes Yes No ES- like cells No Yes No No Yes No genes, mapped genes and DNA markers Genetic information on wild populations The number of inbred... Buffers and media 41 2.4.2 Preparation of embryo extract and fish serum 42 2.4.3 Thawing, freezing and maintenance of cell lines 42 2.4.4 Gene transfer into medaka cell lines and transient expression