Development of a MACS based strategy for isolating rare cell populations from animal tissue for transcription factor studies

DEVELOPMENT OF A MACS-BASED STRATEGY FOR ISOLATING RARE CELL POPULATIONS FROM ANIMAL TISSUE FOR TRANSCRIPTION FACTOR STUDIES MATHIA LEE YU CHUN B.Sc. (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 Page Page i ACKNOWLEDGEMENTS I would like to acknowledge and thank the following valuable contributors to my work: For their valuable support and advice through the years: A.P. Thomas Lufkin (GIS), my thesis advisor; Prof. Edison Liu (GIS) and Prof. Yang Xiaohang (IMCB), members of my thesis advisory committee. For their contribution towards various experiments that were key to the success of this project: Dr. Yap Sook Peng, for her Sox9+/Egfp and Sox9EGFP/EGFP ES cells and technical advice; Valarie Tan, for performing the mice embryo microinjections; Sumantra Chatterjee and Adrian Lim, for performing the zebrafish embryo microinjections; Yang Sun, for technical support for the microarrays; Dr Tamilselvi , for her technical advice and plasmids for the expression of humanized BirA.(All from GIS) For contribution towards the preparation of the thesis and microarray analysis: Dr Michael Gallagher (U.Dublin., Trinity College) . For their help, their lessons, and for simply being there: Members of the Lufkin lab 20059, members of the SK Lim lab 1999-2005, various members of the GIS Stem Cell and Developmental Biology Group. In particular, I would like to thank the following people who have been key to my development as a scientist through these years: Prof .Barry Halliwell, A.P. Lim Sai Kiang, Dr. Yin Yijun,Dr. Michael Gallagher, Rani Ettikan. The following people also provided much needed technical support and advice: Serene Lee, Song Jie, Dr. Clara Cheong, Evangeline, Pierce Lee, Joan Yeo, Dr. Que Jianwen, Dr. Andrew Hutchins, David Wong, Dr Soh Boon Seng and many many more. For their tireless financial, moral and practical support: My parents, Wenlong, Mic For His Plans: My God. Page ii TABLE OF CONTENTS Summary x List of Figures xii Glossary xv The Thesis Chapter Introduction __________________________________ Chapter Background and Literature Review _______________ Section 2.1 Current sample preparation methods are insufficiently representative of in vivo processes studied 2.1.1 Whole organs, tissues or biopsies Section 2.2 Section 2.3 2.1.2 Microdissection 12 2.1.3 Primary cell cultures 16 2.1.4 Established immortal cell lines 18 2.1.5 Fluorescence Activated Cell Sorting (FACS) 21 2.1.6 Survey of recent common sampling methods 24 Magnetic Activated Cell Sorting is suited for low cost, rapid isolation of rare cells from tissue 2.2.1 Clinical applications 26 2.2.2 Research applications 30 2.2.3 Comparison of MACS with other methods 36 Sox9 is an ideal transcription factor for this study because of its biological importance and its known expression patterns 2.3.1 Sox9 as a master regulator of chondrogenesis 43 2.3.2 Sox9 targets, functions and expression patterns 47 2.3.3 Sox9 mutant phenotypes 50 2.3.4 Sox9 dysfunction in human disease conditions 51 Page iii 25 43 Chapter General Methods _______________________________ 54 Section 3.1 Endpoint Polymerase Chain Reaction (PCR) 55 Section 3.2 Western Blotting 56 3.2.1 Protein extraction 56 3.2.2 Bradford Assay 56 3.2.3 3.2.4 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 57 Gel transfer 57 3.2.5 Immunoblotting 57 Section 3.3 Southern Blotting 58 Section 3.4 Cell culture 60 Section 3.5 3.4.1 ES cells 60 3.4.2 HEK293 cells, NIH3T3 cells 61 3.4.3 ATDC5 cells 61 3.4.4 Fluorescence Activated Cell Sorting (FACS) 61 DNA manipulation 63 3.5.1 DNA extraction and purification 63 3.5.2 Restriction Enzyme digestion 64 3.5.3 DNA ligation 64 3.5.4 DNA cloning and amplification in E. coli 64 Page iv Chapter Section 4.1 Section 4.2 Pilot study of MACS using Lngfr: In vitro testing shows samples are highly pure ___________________ 66 Introduction 67 4.1.1 Aims and experimental set up of pilot study 67 4.1.2 Reasons for choice of Miltenyi’s MACS system 68 4.1.3 Reasons for in vitro optimisation 75 Methods 4.2.1 Section 4.3 Section 4.4 77 4.2.2 Expression vector construction: pBAP-EGFP, pBAPEGFP-Lngfr, pMACS Lngfr Neo 77 Transient expression in cells 79 4.2.3 Magnetic Activated Cell Sorting (MACS) 80 Results 81 4.3.1 Overview of experimental approach 81 4.3.2 MACS with HEK293 cells 84 4.3.3 MACS with NIH3T3 cells 87 4.3.4 MACS antibody binding optimisation 92 4.3.5 Cell dissociation optimisation 95 Discussion 99 Page v Chapter Section 5.1 Section 5.2 Section 5.3 Section 5.4 Single step MACS of tissue-dissociated cells: Background levels of non-specifically bound cells are unsatisfactorily high ____________________________ Introduction 104 105 5.1.1 Experimental aim and set up 105 5.1.2 Reasons for the choice of Sox9 108 5.1.3 Homologous recombination in ES cells 109 5.1.4 ES cell lines used: V6.4 and R1 111 5.1.5 MACS with teratomas 112 5.1.6 MACS with wild-type embryos 114 Methods 115 5.2.1 Screening of candidate tags for MACS 115 5.2.2 Dissociation and MACS of cells from tissues 115 5.2.3 Dead Cell Removal 116 5.2.4 Alcian Blue Staining 116 5.2.5 Sox9 targeting construct for Lngfr knock-in 117 5.2.6 Establishing modified ES cell lines 119 5.2.7 Generation of transgenic embryos 5.2.8 Generation of transgenic teratomas 121 5.2.9 MACS isolation of teratoma-dissociated cell samples 122 120 Results 123 +/Lngfr 5.3.1 Generation of Sox9 5.3.2 Generation of Sox9+/Lngfr mouse tissue 127 5.3.3 MACS with teratoma tissue 133 5.3.4 MACS background in embryo tissue 137 5.3.5 Screening of candidate tags for MACS 139 Discussion ES cell lines 123 143 Page vi Chapter Section 6.1 Section 6.2 Section 6.3 Two-step MACS of cells from tissue: significant improvement in sample purity is obtained _________ 150 Introduction 151 6.1.1 Rationale behind the Two-step MACS 6.1.2 Biotin Acceptor Peptide 156 6.1.3 Two-step MACS experimental strategy 159 Methods 162 6.2.1 Construction of pDisplay targeting vectors 162 6.2.2 Construction of pSBL targeting vectors 6.2.3 Construction of hBirA expression vector 6.2.4 X-Gal staining for LacZ 172 6.2.5 MACS using the BAP tag 173 6.2.6 Removal of cell-bound magnetic beads 173 6.2.7 Zebrafish injection and dissociation 173 6.2.8 Confocal imaging 174 167 169 Results 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 Section 6.4 151 175 Optimisation of the two-component surface protein design 175 Gene targeting for the in vivo Two-step MACS: BAP-Lngfr knock in to the Sox9 locus, hBirA knock in to the ROSA26 locus 188 Optimising the Two-Step MACS protocol: 196 Tissue-Dissociation, Magnetic bead removal, Antibody-binding, Surface expression of BAP-Lngfr Two-Step MACS 211 Reservations: Poor NIH3T3 cell viability; Zebrafish developmental abnormality 219 Discussion 230 Page vii Chapter Section 7.1 Section 7.2 Section 7.3 Section 7.4 Transcriptome profiling of MACS-isolated cells: Sox9-related gene expression networks elucidated ___ 240 Introduction 241 7.1.1 Experimental goals and set up 241 7.1.2 Illumina Bead-Chip Arrays 242 7.1.3 Sox9 Transcriptome profiling 245 7.1.4 Limitations of microarrays 248 Methods 250 7.2.1 ATDC5 G418 titration 250 7.2.2 ATDC5 differentiation 251 7.2.3 Generation of ATDC5 stable-transfected lines 251 7.2.4 CAG, CMV, PGK promoter test 251 7.2.5 pCAG-NSox9 and pCAG-CSox9 252 7.2.6 pNBAP- and pCBAP- Sox9 targeting vectors 254 7.2.7 RNA isolation 257 7.2.8 Illumina Bead-Chip Array 258 7.2.9 Microarray Analysis 258 7.2.10 Chromatin Immunoprecipitation 259 Results 262 7.3.1 Microarray of MACS fractions 7.3.2 Elucidation of candidate Sox9-related genes 268 7.3.3 Comparison of data set with existing literature 276 7.3.4 Preparation for in vivo ChIP 7.3.5 Preparation for in vitro ChIP 284 Discussion 262 280 288 Page viii Fernandez-Cancio, M., C. 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Capodieci, Donovan et al 2005) A study by Wong et al and Saito-Hisaminato et al mapped the transcription profiles of several human Page 8 organs including liver, lung, brain and placenta in order to establish a database of tissuespecific gene profiles in a normal healthy state, against which future studies could compare as a reference point (Wong, Hafeman et al 2003) (Saito-Hisaminato, Katagiri et al 2002)... commercially prepared RNA from whole tissues saves the laboratory a lot of time Commercial preparations are also used when animal facilities are unavailable In other cases, commercial preparations of human tissue RNA circumvent the Page 10 need to gain the approval of ethics committees However, they do have an impact on the experimental findings ATP-binding cassette transporters serve a transport and regulator... Detection of cartilage tissue in teratoma 131 5.3.2.3 The feasibility of using transgenic teratoma tissue to develop a cell isolation strategy from complex tissue was established 132 5.3.3.1 MACS samples collected for transcriptome analysis by Illimina microarrays 135 5.3.3.2 % of anti-Lngfr-FITC labelled cells in the various MACS fractions 136 Page xii 5.3.4.1 Enrichment of very rare cells (~0.1%) from. .. explain and describe how we developed and validated our cell isolation strategy Through pilot studies with cell cultures, we ensured that MACS, by targeting cells expressing a transgenic cell surface Lngfr protein, was capable of isolating the desired cells to a satisfactory level of sample quantity, purity and recovery (Chapter 4) We then applied this strategy to animal tissues Using transgenic teratomas... studies utilizing MACS In my evaluation of the sampling methods, I am taking into account the requirements of both human and animal studies and will make comparisons against MACS samples where appropriate I will not be surveying plant or microbial studies 2.1.1 Whole organs, tissues or biopsies Animal tissue samples are sometimes used in place of, or to validate cell culture models as they are thought to... (Ishibashi, Hanyu et al 2003),between benign and malignant prostate tumors (Kube, Savci-Heijink et al 2007) and between recurring and no-reccuring prostate cancers (Nariculam, Freeman et al 2009) Sugiyama et al demonstrated through gene profiling that whole cancer tissue contains a significant proportion of normal cells that interfere with accurate profiling of cancer cells and that at least some form of. .. be the transcription profile of that particular cell type in the normal state, rather than the profile of the entire organ Having samples consisting purely of cells of interest is especially relevant for developmentally regulated processes where each cell is spatio-temporally regulated by its microenvironment, and each cell has a specific fate which differs from other cells Tissues are made up of closely... gene of interest so that only cells expressing this gene will be isolated by the Two-step MACS Our method addresses the current dearth in cell isolation methods that can purify rare cells, expressing a specific gene of interest, from complex animal tissue in a way that is fast, affordable and can be scaled up to give sufficient sample quantities Our aim is to develop such a cell isolation strategy that... chemical methods Laser capture microdissection (LCM) has been used on a wide variety of tissues, including Langerhans cells (McClain, Cai et al 2005), hepatocytes from liver (Marko-Varga, Berglund et al 2003), hypothalamic neurons (Paulsen, Larsen et al 2009), intestinal epithelial cells (George, Wehkamp et al 2008) and experimentally induced cerebral aneurysms in rat (Aoki, Kataoka et al 2008) Trophoblasts... cells of interest; b) getting sufficient quantities of these cells Current methods used for isolating specific cell populations from tissues are either too costly or technically challenging to obtain large quantities of samples, or do not yield sufficiently pure samples We addressed these challenges by developing a Magnetic Activated Cell Sorting (MACS) -based strategy to isolate rare cells of interest from . that rare cell populations can be isolated to sufficient purity for sensitive downstream assays. We termed this strategy the „Two-step MACS , and showed that a rare population of cells (~1% of. Screening of candidate tags for MACS 115 5.2.2 Dissociation and MACS of cells from tissues 115 5.2.3 Dead Cell Removal 116 5.2.4 Alcian Blue Staining 116 5.2.5 Sox9 targeting. which transgenic animal tissue can be derived. Sox9 was chosen as our model transcription factor of interest for its biological importance as the master regulator of chondrogenesis and for its