Transgenic Vectors Core

INTRODUCTION

Neuromuscular Junction - Josh Sanes

Mouse transgenesis, the stable integration of foreign DNA into the mouse genome, is a key approach used in biomedical research to explore gene function and regulation and to model human disease. Applications include gain or loss of function studies, mapping regulatory elements or functional domains by analysis of gene expression and subcellular localization patterns, and generating reporter lines for in-vivo imaging of neuronal projection patterns. Innovations in mouse transgenesis technologies include conditional gene modifications that inactivate gene function in tissue-specific manners, thereby overcoming embryonic lethality; and BAC transgenes to circumvent the position effects commonly found in standard transgenic mice.

The Transgenic Vectors Core uses recombineering technologies to generate complex gene modifications not possible with restriction enzymes, instead using PCR and homologous recombination in bacteria to modify DNA. Phage-based homologous recombination systems in E. coli have made it possible to introduce mutations anywhere in the genome. Large segments of genomic DNA, such as those found on BACs (Bacterial Artificial Chromosomes) are now routinely modified using recombineering technologies. This facilitates production of mutant mice that were previously impossible to generate.

The Transgenic Vectors Core designs and constructs mouse transgenes and gene targeting vectors (including conditional knock-outs and reporters, and BAC transgenes) on a fee-for-service basis and is available for use by all Washington University investigators. Services are also available to users outside of the Washington University community.

SERVICES

Retina. Rachel Wong

PROMOTER TRANSGENES

Promoter transgenes drive expression of a reporter or other gene of interest in a tissue or cell-type specific manner. Promoter transgenes integrate in random positions within the genome.

KNOCK-OUT AND KNOCK-IN GENE TARGETING VECTORS

Gene targeting vectors introduce mutations into endogenous loci by homologous recombination in ES cells. Mutation strategies include deletion of functional domains or translational start sites, introduction of frame shifts that terminate translation, deletion of entire genes, or insertion of reporters.

CONDITIONAL PROMOTER TRANSGENES AND GENE TARGETING VECTORS

Conditional gene modifications use the site-specific recombinase systems Cre-loxP or Flp-FRT to irreversibly inactivate (or activate) gene function in tissue or cell-type specific manners. Conditional approaches overcome the embryonic lethality associated with some germline null mutations or over-expression of a gene of interest. Gene expression is controlled by the interaction of Cre or Flp recombinase with a “floxed” or “frted” target gene using a dual transgenic mouse system.

BAC TRANSGENES

Host sequences surrounding the transgene integration site can modify transgene expression; causing it to be weak, undetectable, or expressed in only a subset of expected cells or tissues. BAC transgenes can be used to ensure position-independent expression of the gene of interest by providing all regulatory sequences needed on a large (~150 – 200kb) segment of genomic DNA.

CUSTOM SERVICES

Custom services such as site-directed mutagenesis, protein expression vectors, and zebrafish transgenes are also available. Please inquire.

CONSTRUCT REQUEST

Synaptic Vesicles, Rachel Wong

An initial consultation with the principal investigator is arranged to discuss the project strategy, produce a general design of the construct, and identify needed DNA fragments. After approval of the final construct design by the investigator and completion of a project submission form, the core will construct the vector, confirm the construction is correct by DNA sequencing, and prepare the dna for microinjection or ES cell electroporation. Construct requests will be filled in the order received with an estimated turn-around time of approximately 2 - 3 months depending upon the complexity of the project.

STAFF

Neuromuscular junction - Josh Sanes

Renate Lewis
Staff Scientist
lewisr@neuro.wustl.edu
314-362-5224

Faculty Oversight:

Dr. Joy Snider
Dr. Karen O’Malley
Dr. Naren Ramanan

SUPPORT

ChAT KO NMJ Synaptic Sites - Josh Sanes

The Transgenic Vectors Core is supported by The Hope Center for Neurological Disorders, The Department of Neurology, The McDonnell Center for Cellular and Molecular Neuroscience, and The McDonnell Center for Systems Neuroscience.

REFERENCES

Bipolar Cells, Rachel Wong
Branda CS, Dymecki SM. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell. 2004 Jan;6(1):7-28. Review.
Copeland NG, Jenkins NA, Court DL. Recombineering: A Powerful New Tool for Mouse Functional Genomics. Nature Genetics. 2001 2: 769-779.

Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 2000 28(1): 41-51.

Hatten ME, Heintz N. Large-scale genomic approaches to brain development and circuitry. Annu Rev Neurosci. 2005 28:89-108.

Heintz N. Analysis of mammalian central nervous system gene expression and function using bacterial artificial chromosome-mediated transgenesis. Human Molecular Genetics. 2000 9(6): 937-943.

Kucharczuk KL, Love CM, Dougherty NM, Goldhamer DJ. Fine-scale transgenic mapping of the MyoD core enhancer: MyoD is regulated by distinct but overlapping mechanisms in myotomal and non-myotomal muscle lineages. Development. 1999 126(9): 1957-65.

Liu P, Jenkins N, Copeland NG. A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations. Genome Research. 2003 13:476-484.

Misgeld T, Burgess RW, Lewis RM, Cunningham JM, Lichtman JW, Sanes JR. Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron. 2004 36(4): 635-48.

Sasazuki T, Sawada T, Sakon S, Kitamura T, Kishi T, Okazaki T, Katano M, Tanaka M, Watanabe M, Yagita H, Okumura K, Nakano H. Identification of a novel transcriptional activator, BSAC, by a functional cloning to inhibit tumor necrosis factor-induced cell death. J Biol Chem. 2002 277(32): 28853-60.

van der Neut R. Targeted gene disruption: applications in neurobiology.J Neurosci Methods. 1997 71(1):19-27.

Yoon K, Gaiano N. Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci. 2005 8(6):709-15.

LINKS

Washington University Mouse Genetics Core

Siteman Cancer Center Murine Embryonic Stem Cell Core

Department of Pathology and Immunology Microinjection Core

Washington University Core Facilities

Hope Center for Neurological Disorders

Department of Neurology

Department of Anatomy and Neurobiology