VTMI 664

Strategies for Manipulating the Mouse Genome

Fall Semesters, Even-numbered Years

Professor: Dr. Danna B. Zimmer

Associate Professor of Veterinary Pathobiology

Office: Veterinary Medical Administration Building (#1026), Room 220

E-mail: dzimmer@cvm.tamu.edu

Voice: 979-845-8040

Office Hours: Students are encouraged to contact the instructor at any time via telephone, e-mail, and/or pre-arranged appointments.

Class Meetings: Two 1½ hour sessions per week, time and location TBA

Course Overview and Objectives:

I believe that effective graduate teaching is much more than conveying factual information. There is never enough time to teach you all the information you will need to know; some of what I teach you will change in the coming years; and there will always be new things to learn. Therefore, my objective is to provide you with the basic concepts/skills needed to navigate the scientific literature and effectively communicate with other scientists working in the field. To achieve these objectives, information is presented in an interactive manner and active/thoughtful student participation is an integral part of my courses. Basic concepts are presented via discussion of seminal papers and the current/future directions of the field are conveyed through in-depth discussions of the primary scientific literature. Understanding, utilization, and communication of basic concepts are evaluated via Critical Thinking Exercises and hypothetical research problems.

The overall objective of this course is to provide you with the basic concepts and skills needed to design/generate constructs for mouse genome modification and to evaluate scientific literature utilizing genetically modified mouse models. Primary scientific literature from diverse fields including genetics, toxicology, neuroscience, infectious diseases, developmental biology, gene expression, and pharmacology will be used to illustrate the different technologies. Basic concepts will be acquired through your evaluation/class discussion of this body of literature. A series of Critical Thinking Exercises (CTE’s) will be used to assess your comprehension of these basic concepts. An integrative research proposal will allow you to apply fundamental concepts and critical thinking skills to a hypothetical project. The CTEs represent key milestones in the process of de novo development of a genetically modified mouse models and will be instrumental in the timely development of a thought provoking and scientifically sound research proposal. You will also actively participate in both self- and peer-review of assignments.

The course is designed to improve your competencies in the following areas:

1. Evaluation of genetically modified mouse models. You will acquire the basic concepts and working vocabulary needed for the analysis/evaluation of genetically modified mouse models. Class participation, Literature Evaluations, and Critical Thinking Exercises will be indicative of your progress in this area.

2. De novo generation of constructs for mouse genome modification. Using the basic concepts learned throughout the course, you will generate a new mouse model that can be used to address the hypothesis/question being asked/tested in your thesis/dissertation proposal. The Research Proposal will be used to evaluate your progress in this area.

3. The peer-review process. You will evaluate the work of others and provide constructive comments/criticisms regarding their work. You will also receive analyze, interpret, and integrate into your own work constructive criticism from your peers. Class participation, Literature Evaluations, and Critical Thinking Exercises will be indicators of your progress in this area.

4. Scientific communication. You will enhance your ability to write clearly, listen carefully and speak in a professional and effective manner. Class participation, Literature Evaluations, Critical Thinking Exercises, and the Research Proposal will measure your performance in this area.

Textbook and Recommended Materials:

Because the course will be taught from the primary literature there is no required textbook. The following textbooks are recommended for reference:

1. Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer. Manipulating the Mouse Embryo: A Laboratory Animal, 3^rd Edition. New York, Cold Spring Harbor Press, 2003

2. I.J. Jackson and C.M. Abbot. Mouse Genetics and Transgenics: A Practical Approach. Oxford, Oxford University Press, 2000.

3. Carl Pinkert. Transgenic Animal Technology: A Laboratory Handbook, 2^nd Edition. New York, Academic Press, 2002.

4. Joyner, A. L., editor. Gene Targeting, a Practical Approach. New York, Oxford Univeristy Press, 1999.

5. Houdebine, L.-M. Animal Transgenesis and Cloning. New Jersey, John Wiley & Sons, Inc., 2003.

Additional information can be found at the following websites:

Proposal forms and evaluation instructions:

http://www.nih.gov

http://grants1.nih.gov/grants/funding/phs398/phs398.html

http://www.csr.nih.gov/CDG/CD%20Guidelines/r01.pdf

Literature databases:

PubMed database: http://www.ncbi.nlm.nih.gov

Ovid at TAMU Library Electronic Resources: http://library.tamu.edu

DNA Sequence Databases :

Entrez and Blast: http://www.ncbi.nlm.nih.gov

Mutant Mouse Resources:

http://www.jax.org

Prerequisite:

Registrants must be formally enrolled in a graduate program (M.S. or Ph.D.) at Texas A & M University.

Instructional Activities:

A diverse compliment of instructional activities will accompany the interactive presentation of the material. Each instructional activity represents an important milestone in your progress towards the overall course objective: evaluation and de novo generation of constructs for mouse genome modification. Daily Class Participation and Literature Evaluations emphasize comprehension, analysis, and evaluation of fundamental concepts. The Critical Thinking Exercises and Research Proposal emphasize synthesis and application of basic concepts.

1. Class Participation (15%): Class participation is a significant portion of the course grade and should be given the same priority as the other grade components. To excel in this area, you should strive to share high quality comments every class session that extends the discussion in meaningful ways. This requires some knowledge of (i.e. having read) the papers that will be discussed during each class session, even unassigned papers. Low quality comments and participating just to participate will negatively impact your class participation grade. You should note that attendance alone does not help your performance in the class participation area. Two mechanisms will be used to evaluate class participation: a Class Participation Evaluation Form and a series of One-Minute Papers. These two scores will be averaged and multiplied by X 0.15 to obtain the class participation component of your final grade.

a. Class Participation Evaluation Form: You will be asked to complete a Class Participation Form (Appendix Item 3) during and at the completion of the class. This form asks you to evaluate all aspects of your class participation and to assign a grade. In completing this form you will need to justify your grade by providing illustrations that support your points. To do this, it is suggested that you document the nature and quality of your contributions. I will collect these forms, read them carefully, make comments and return them to you with a participation grade. The average grade on these two forms represents one-half of your class participation grade.

b. One-Minute Papers: One-Minute Papers will be an integral part of the course. These papers will assist you in developing the scientific question(s) and experimental strategies needed for your Research Proposal. Equally important is the instant feedback that these exercises will provide regarding your progress in the course. One-Minute Papers are writing to learn exercises and grading will emphasize completeness and depth of the thoughts recorded, not presentation. However, your comments must be clear and legible. The average grade on the One-Minute Papers represents one-half of your class participation grade.

2. Literature Evaluations (35%): You will routinely read and evaluate selected articles from the primary scientific literature. Because each article that you read/evaluate will improve your literature evaluation skills, it is recommended that you read/evaluate all of the articles cited in the Literature List for the course. This is not as daunting a task as it may seem because our goal is evaluation of the construct(s) used to modify the genome, not necessarily the entire article. To excel in this area you should strive to not only understand the basic concepts presented in the article but to relate the findings in the article to the course’s learning objectives and your own research proposal. This goal can be better achieved by reading/working on the papers/evaluations multiple days rather than completing the tasks in a single setting. Session 2 of the course will be devoted to the development of a Literature Evaluation Form. You will be required to complete a total of 10 Literature Evaluation Forms. Articles for LE1, LE2, LE4, LE5, LE7, LE9, and LE10 will be assigned by the instructor during the class session prior to their due dates. Acquisition of copies of the articles is your responsibility. You will select the articles for the three class sessions entitled Journal Club, (LE3, LE6, and LE8). Your article must include the generation of a new genetically modified mouse model, be pertinent to your Research Proposal, and be approved by the instructor no later than two class sessions before the LE due date. Students will be required to bring 2 copies of the completed Literature Evaluation form to class—one copy will be turned into the instructor before class begins and the other copy will be used in class to facilitate discussion. Your average grade on the ten Literature Evaluations X 0.35 will be the Literature Evaluation Component of your final course grade.

3. Critical Thinking Exercises (35%): A series of Critical Thinking Exercises (CTEs) will be used to assess your mastery and utilization of basic concepts. The CTEs represent key milestones in the design/generation of DNA constructs for development of genetically modified mouse models. As such, these exercises will provide the foundation you need to efficiently develop a thought provoking and scientifically sound research proposal. Unlike the One-Minute Papers, CTEs are learning to write exercises and will be graded on organization, spelling and grammar as well as content. Each CTE will graded on a 100 point scale and the average grade of the 6 CTES x 0.35 will constitute the Critical Thinking Exercise component of your final grade.

CTE-1—Literature Search of Relevant Genetically Modified Mouse Models, Due Session 6: This exercise requires that you summarize the available genetically modified mouse models pertinent to your research proposal and indicate the critical need that your proposed model will meet. This information should be presented in paragraph form (400 words or less) and a bibliography should be attached. Pertinent search results should be included as Appendices.

CTE-2—Identification of DNA Sequences for Construct Generation, Due Session 15: Generation of a construct for genome modification requires both DNA sequences and restriction maps. Using the databases at the National Center for Biotechnology, and any other needed resources, you will identify the DNA sequences (cDNA and/or genomic DNA clones) that will be needed to generate your construct. Your findings should be summarized in a single paragraph (300 words or less). Pertinent DNA sequences and restriction maps should be attached as Appendices.

CTE-3—Grant Proposal Review, Due Session 16: You will be assigned a R03 grant application to review. In order to focus our discussion on research proposal organization and evaluation, I have selected a proposal that does not utilize genetically modified mouse models. The proposal will be distributed at the end of session 14 and, as a group, we will review aspects of proposal development/review unrelated to scientific merit. Prior to the next class session, you will use the Guide for Assigned Reviewers’ Preliminary Comments on Research Grant Proposals (Appendix Item #2) to evaluate the proposal’s scientific merit and assign a priority score. Duplicate hardcopies of your review are due at the beginning of class session 16. Your review will be graded on completeness and accuracy.

CTE-4—Research Proposal Abstract, Due Session 18: In this exercise you will generate the abstract for your research proposal. Unlike the research proposal, abstracts are often written at the last minute with little attention to detail. However, the abstract is one of the most important parts of a proposal/paper. Your abstract must be submitted on PHS 398 Form Page 2 and adhere to the formatting instructions in the PHS 398 Instructions (Appendix Item #1). Be sure that you use the most recent version of these forms. Your abstract should describe the problem that will be addressed, the approach that will be used, the significance of the problem, and the innovation of the proposal. Your abstract will be graded on completeness, clarity, and adherence to the formatting instructions. You need to bring 10 hardcopies of your abstract to class session 18.

CTE-5—Peer-Review of Research Proposal Abstracts, Due Session 19:

You will read the abstracts submitted by the other class participants and comment on the significance, approach, and innovation of the proposals. These comments should be brief and in bulleted format. You will also be asked to assign a score to the abstracts using the following range: 1—outstanding/highest priority; 2- excellent/high priority; 3-average/medium priority; 4-below average/low priority; 5-unacceptable/lowest priority.

CTE-6--Peer-Review of Research Proposals, Due Date TBA: You will read, review, and score the Research Proposal of a fellow classmate using the Critique and Overall Evaluation sections of the NIH Guide for Assigned Reviewers’ Preliminary Comments on Research Grant Proposals (Appendix Item #2). Your review will be graded on completeness, accuracy, and constructiveness of the comments.

4. Research Project Proposal (35%), Due Session 26: An integrative research proposal will allow you to demonstrate mastery and utilization of basic concepts. The research report will describe the generation and initial characterization of a new genetically modified mouse model that can be used to test the hypothesis(s) proposed in the student’s thesis/dissertation project. To excel at this task you will need to integrate all of the skills that have been developed during the course: construct design, critical thinking, scientific writing, and evaluation of scientific literature/proposals. This is a learning to write exercise and your grade will reflect organization, spelling, and grammar as well as content. The organization and format of the report should follow the NIH PHS 398 guidelines, with the exception that the total length of the research plan (items A-D) is limited to eight pages. The proposal must include Form Page 1: Face Page; Form Page 2: Description/Performance Sites; Form Page 3: Table of Contents; Biographical Sketch; and the Research Plan. Your research proposal is due at the beginning of session 25. Proposals will be evaluated on scientific merit (80%) per the NIH Guide for Assigned Reviewers’ Preliminary Comments (Appendix Item 2), written presentation (10%), and oral presentation (10%; per Appendix Item 4). The Research Proposal Score that will be used in calculating your final grade will be this score X 0.35.

Grading:

The final course grade will be based on 4 major components. These components and their weights are listed below.

1. Critical Thinking Exercises (6 total) 35%

2. Research Proposal 35%

3. Literature Evaluations 15%

4. Class Participation (One-Minute Papers/Evaluation Form) 15%

Numerical point totals will be converted to letter grades on a straight >90 (A), >80 (B), >70 (C), >60 (D), <60 (F) continuum. The criteria for each component are detailed in the following subsections.

Make-Ups, Late Assignments:

All assignments are to be turned in prior to the start of class on the due date listed in Assignment List (pages 9-10). There will be no make-ups and late assignments will be accepted only in the case of University excused absences. In order to receive credit, all assignments must be submitted to Dr. Zimmer prior to the start of class. If you are unable to attend class, the only fail-safe mechanism for submission of an assignment is delivery of a hardcopy directly to Dr. Zimmer or the secretary in VMA 201 prior to class. A grade of 0 will be assigned for any One-Minute Papers that are missed.

Academic Honesty:

The Aggie Honor Code states:

“An Aggie does not lie, cheat, or steal, or tolerate those who do.”

Upon accepting admission to Texas A & M University, individuals immediately assume a commitment to uphold the Honor Code, to accept responsibility for learning, and to follow the philosophy and rules of the Honor System. Ignorance of the rules does not exclude any member of the Texas A & M University community from the requirements or the process of the Honor System. For additional information visit: http://www.tamu.edu/aggiehonor/.

Services for Students with Disabilities:

The Americans with Disabilities Act (ADA) is a federal anti-discrimination statute that provides comprehensive civil rights protection for persons with disabilities. Among other things this legislation requires that all students with disabilities be guaranteed a learning environment that provides for reasonable accommodation of their disabilities. If you believe you have a disability requiring an accommodation, please contact the Department of Student Life, Services for Students with Disabilities, in Room 126 of the Koldus Building or call 845-1637. Please also contact Dr. Zimmer within the first two weeks of class.

Biographical Sketch: Dr. Danna Zimmer

I am an Associate Professor in the Department of Veterinary Pathobiology and serve as Director of the Embryonic Stem Cell Core in the Texas A & M System Genetically Engineered Mouse Facility. I was raised in Houston, Texas and received my B.A. degree from Rice University with a double major in Biochemistry and Biology. I received a Ph.D. degree in Cell Biology from Baylor College of Medicine and did post-doctoral studies at Baylor College of Medicine and Vanderbilt University. As a graduate student and post-doctoral fellow, I studied protein structure-function relationships, protein-protein interactions, and macromolecular assemblies. My first faculty position was in the Department of Pharmacology in the College of Medicine at the University of South Alabama in Mobile, AL where I taught graduate/medical students, served as major advisor for Ph.D. students/post-doctoral fellows, and ran an independent NIH-funded research program. I became interested in S100 proteins when I was a post-doctoral fellow and have continued to study this large family of intra- and intercellular calcium-binding proteins in my own laboratory. We use a multidisciplinary approach to delineate S100-mediated signal transduction cascades in the nervous system and to determine how these pathways contribute to neurological diseases and cancers.

It was during my tenure at Vanderbilt University that I was first introduced to genetically modified mouse models, specifically transgenic mice. I provided the immunohistochemical expertise for a project in which expression of a human growth hormone reporter gene was being used to identify regulatory elements in the promoter of the phosphenolpyruvate carboxykinase gene, an enzyme implicated in diabetes. As the technology advanced, I became interested in using genetically modified mouse models to study S100 proteins. During a sabbatical in the laboratories of Dr. Robert Schwartz and Francesco DeMayo, I generated what would be the first in a series of mice with genome modifications in S100 encoding loci as well as several NKX transcription factor loci. During my sabbatical I was able to watch and perform all of the tasks associated with the generation of genetically modified mouse models: construct design/generation, embryonic stem cell targeting, mouse colony management, and DNA/embryonic stem cell microinjection. When I returned to the University of South Alabama, I set-up and served as Director of the Transgenic Mouse/Embryonic Stem Cell Core Laboratory in the College of Medicine. As part of the core’s activities, I taught a graduate course in Genetically Modified Mouse Models for Biomedical Research and laboratories in microinjection techniques. This course focuses on the design/generation of the constructs used to modify the mouse genome. As part of the educational component of the Texas A & M System Genetically Engineered Mouse Facility, it is open to all graduate students.

Topics/Assignments

Session	Discussion Topic	Assignment(s) Due
Session 1	Mouse Genetics/Nomenclature Construct Building Blocks
Session 2	Transgene Expression
Session 3	Factors that Influence Construct Design/Generation
Session 4	Factors Influencing Construct Design/Generation
Session 5	Transgenic Constructs/Technology
Session 6	Transgenic Constructs/Technology (continued)	CTE1--Literature Searches Class Participation Form
Session 7	Transgenic Constructs/Technology (continued)	LE1
Session 8	Transgenic Constructs/Technology (continued)	LE2
Session 9	Transgenic Constructs/Technology (continued)	LE3—Journal Club Selection
Session 10	Journal Club
Session 11	ES-Cell Technology
Session 12	ES-Cell Technology (continued)	LE4
Session 13	ES-Cell Technology (continued)
Session 14	ES-Cell Technology (continued)	LE5
Session 15	ES-Cell Technology (continued) CTE-4 Introduction	CTE2--Identification DNA Sequences
Session 16	Grant Proposal Review	CTE3--Grant Proposal Review
Session 17	Journal Club	LE6—Journal Club Selection
Session 18	Site-Specific Recombinases	CTE4--Research Proposal Abstract LE7
Session 19	Review of Proposal Abstracts	CTE5--Abstract Peer-Review
Session 20	Inducible Systems	LE8--Journal Club Selection
Session 21	Journal Club
Session 22	Toxicology Models	LE9
Session 23	Humanized Mouse Models
Session 24	siRNA	LE10
Session 25	Genome-Wide Modifications
Session 26	Genome-Wide Modifications (cont.)	Research Proposal
Session 27	Presentation of Research Proposals
Session 28	Presentation of Research Proposals
TBA		CTE6 Research Proposal Reviews Class Participation Form