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Building a Cassette-Based System for Combined Eukaryotic Gene Expression

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Building a Cassette-Based System for Combined Eukaryotic Gene Expression AN ABSTRACT OF THE THESIS OF Raviteja Madhira for the degree of Honors Baccalaureate of Science in Biochemistry and Biophysics presented on August 26, 2009. Title: Building a cassette-based system for combined eukaryotic gene expression. Abstract Approved:________________________________________________________ Michael K. Gross Gene regulatory networks control the state of a cell. Models of such networks have been produced for sea urchin development and suggest that cell type may be defined by distinct network kernels. Such kernels are composed of a set of sequence specific DNA-binding transcription factors (SSTFs) that maintain each other’s expression through cell type-specific cis- regulatory modules. Ectopic expression of some individual SSTFs leads to trans-differentiation, or the conversion of one cell type to another. Our hypothesis is that trans-differentiation will be more efficient and targeted if specific SSTF combinations, corresponding to those of a network kernel, are expressed ectopically. A transient, polycistronic, cassette-based expression system was designed and built. This system allow for Lbx1-dependent network kernels to be transiently introduced into heterologous cells. Transfecting a cell with a specific combination of SSTFs may trigger establishment of the corresponding endogenous network kernel and thereby lead to trans- differentiation. Five SSTF ORFs (Lbx1, Lmx1b, Pax2, Pax8, and Isl1) were PCR amplified with gene-specific primers that included AsiSI and PacI restriction sites. A base bacterial vector was created with a custom polylinker to receive the PCR-generated minimal ORFs. This custom polylinker was constructed so that PCR-generated minimal ORFs could readily be transferred to other vector systems by classic recombinant methods. The AsiSI/PacI minimal ORF fragments are transferred to a pcDNA 575/576 mammalian expression vector containing the IRES ORF cassette to generate IRES-ORF or ORF-IRES expression cassettes. Polycistronic expression cartridges of fluorescent proteins and SSTF kernels need to be completed before functional tests can be done. Keywords: Network Kernel, Network Model, SSTFs, Lbx1, Trans-differentiation Corresponding e-mail address: [email protected] Building a cassette-based system for combined eukaryotic gene expression by Raviteja Madhira A PROJECT submitted to Oregon State University University Honors College In partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biochemistry and Biophysics Presented August 26, 2009 Commencement June, 2010 Honors Baccalaureate of Science in Biochemistry and Biophysics project of Raviteja Madhira presented on August 26, 2009. APPROVED: ________________________________________________________________________ Mentor, representing Biochemistry/Biophysics ________________________________________________________________________ Committee Member, representing Biochemistry/Biophysics ________________________________________________________________________ Committee Member, representing Pharmacy ______________________________________________________________________ Dean, University Honors College I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request. ________________________________________________________________________ Raviteja Madhira, author Acknowledgements I sincerely thank Dr. Michael Gross for giving me an opportunity to work in his research lab, for spending enormous amounts of time teaching me various lessons, and for being a caring and a concerned person. I also thank Jun Xu for his valuable support in the lab. Finally, I thank Dr. Victor Hsu and Dr. Chrissa Kioussi for agreeing to be my committee members. TABLE OF CONTENTS Page INTRODUCTION……………………………………………………………..1 Network Kernels……………………………………………………….. 1 Design of a Transient Coexpression System…………………………... 4 RESULTS……………………………………………………………………...8 Vector to Receive Open Reading Frame Cassettes…………………….. 8 PCR Amplification of the Gene Cassettes……………………………... 16 Cloning the PCR Products in BS 531/534 Vector…....………………… 18 Creating ORF-IRES Cassette in a Mammalian Expression Vector……. 23 Insertion of IRES Cassette to pcDNA 575/576 Vector………………… 24 Building ORF-IRES or IRES-ORF Cassettes in pcDNA-IRES……….. 25 METHODS AND MATERIALS……………………………………………...26 PCR Amplification…………………………………………………….. 26 Preparation of PCR Amplified Fragments for Ligation………………... 27 Ligation and Chemical Transformation………………………………... 28 Alkaline Lysis Minipreps………………………………………………. 29 DISCUSSION……………….. ……………………………………………….30 Adapting Other Vector Systems to Accept ORF Cassettes……………. 30 Amplification of ORFs………………………………………………… 34 Difficulty in Obtaining Minimal ORF Clones…………………………. 38 Future Studies………………………………………………………….. 42 BIBLIOGRAPHY……………………………………………………………...44 LIST OF FIGURES Figure Page 1. Possible Lbx1-dependent Network Kernels………………………………... 4 2. Amplification of Minimal ORFs…………………………………………… 6 3. Design of the Transient Coexpression Vector……………………………... 7 4. Prevalence of Internal Restriction Sites in Mouse Coding Sequences…….. 9 5. Rationale for Placement of Sites at the Start and Stop Ends of ORFs……... 10 6. Sets of Isocaudomer Sites in the Designed Polylinker…………………….. 11 7. Sequence of the Final Polylinker in Base Vector………………………….. 12 8. Sequence of the First Oligomer Inserted into BS KS+……………………... 12 9. Restriction Analysis of BS 531/532 Clones ………………………………. 13 10. Restriction Map of BS 531/532…………...……………………………….. 13 11. Sequence of the Second Oligomer Cloned into BS 531/532………………. 15 12. Restriction Analysis of BS 531/534 Clones ………………………………. 15 13. Restriction Map of BS 531/534……………………………………………..15 14. Restriction Analysis of BS 531/534 Clone 7……......……………………... 16 15. Gene-Specific Primers to Amplify ORFs………………………………….. 17 16. PCR Amplified ORFs ………………...…………………………………… 18 17. Excised Band of BS 531/534………………………………………………. 19 18. Restriction Analysis of Plasmid Clones……………………………………. 20 19. Base Vectors Bearing Minimal ORF Cassettes……………………………. 22 20. Custom MCS to Replace MCS of pcDNA 3.1(+) Vector………………….. 23 21. Restriction Map of pcDNA 575/576……………………………………….. 24 LIST OF FIGURES (Continued) Figure Page 22. PCR Amplification of IRES……………………………………………….. 24 23. Restriction Map of pcDNA-IRES…………………………………………. 25 24. Redesigning a Bacterial Expression Vector to Receive ORF Cassettes……………………………………………………………… 31 25. Redesigning a Yeast Expression Vector to Receive ORF Cassettes……………………………………………………………… 32 26. Redesigning a Mammalian Expression Vector to Receive ORF Cassettes……………………………………………………………… 32 27. Identifying the Correct Annealing Temperature for the Lbx1 ORF…………………………………………………………... 37 28. Typical Range Finding Experiment to Optimize [Mg+2] and Annealing Temperature……………………………………………………. 38 LIST OF TABLES Table Page 1. EGFP ORF Amplification…………………………………………………. 26 2. IRES ORF Amplification………………………………………………….. 26 3. dsRed ORF Amplification…………………………………………………. 26 4. Pax8 ORF Amplification…………………………………………………... 26 5. Lmx1b ORF Amplification………………………………………………… 27 6. Pax2 ORF Amplification…………………………………………………... 27 7. Isl1 ORF Amplification……………………………………………………. 27 8. Lbx1 ORF Amplification…………………………………………………... 27 9. %GC and Tm Analyses of ORF Primer Pairs………………………………. 35 10. Transformation Analysis of BS-ORF Clones……………………………… 39 11. Error Rate of Taq Polymerase for Different Assays……………………….. 41 To my parents Introduction Network Kernels Extensive research to understand the development of sea urchin endomesoderm led to the development of network models1-3. Network models are used to represent the highly sophisticated circuits of regulatory interactions among transcription factors and their genes that guide development4, 5. Within the sea urchin regulatory network, researchers have identified ‘evolutionarily inflexible sub-circuits’ that were named network kernels4. Network kernels consist of combinations of 5-10 sequence specific DNA-binding transcription factors (SSTFs) that maintain each other’s expression and thereby define a stable cell type. Our underlying hypothesis is that cell types, such as hepatocytes, epithelial cells, keratinocytes, or fibroblasts, are each defined by a distinct and unique network kernel. When a change in cell type occurs, the established network kernel of that cell type is destabilized and another network kernel forms. The recent success at reprogramming fibroblast cultures to become embryonic stem (ES) cells is consistent with the idea that SSTFs network kernels specify cell type. Researchers introduced different combinations of SSTFs into primary fibroblast or other somatic cell lines to create induced pluripotent cells. They tried many different combinations of SSTFs. However, they selected SSTFs that are normally expressed and play essential roles in the development of ES cells. Expression of a specific combination of four SSTFs, such as Oct3/4, Sox2, c-Myc, and Klf4, from retroviral expression vectors caused infected cells to obtain expression profiles that were more similar to ES cells than to the original cell types6, 7. We infer that these SSTFs constituted key components of the network kernel that specifies ES cells. Artificially introducing this network kernel or a key subset of it triggered the establishment of the endogenous network kernel that is normally stable in ES cells. If this inference is correct, then it suggests the possibility that one cell type
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