This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The development of methods for the simple and accurate manipulation of Bacterial Artificial Chromosomes (BACs) in my laboratory has allowed the utilization of an alternative and highly efficient strategy for analysis of CNS specific genes (Heintz 2000). This approach is based on two simple facts: large genomic DNA fragments (100KB) are in most instances expressed independent of the site of integration into the genome of transgenic mice;inclusion of epitope tags and marker proteins into endogenous loci of invertebrate genes has in most cases not altered the patterns of expression of these genes or the localization of their encoded products within the cell. To take advantage of this information, a homologous recombination system was established in E. coli that allows for preparation of BACs with highly precise modifications. Using this system, it is possible to create mutations in BACs that range from single nucleotide changes to deletions of tens of kilobases to insertions of marker genes of several kilobases. One can, therefore, construct BACs that allow very rapid analysis of the expression pattern of the gene of interest, the localization of its encoded product, high-resolution visualization of the morphology of cells expressing the gene, and determination of the projection patterns of these cells. Mice made using these techniques also carry epitope tagged proteins that can be used for affinity purification of complexes carrying the protein of interest. The use of epitope tags for determination of the subcellular distribution of proteins in invertebrates and in cultured mammalian cells is very well established. Because of the precision of homologous recombination in E. coli, it is quite simple to introduce an epitope tag into the protein encoded by the gene of interest in the BAC at the same time that one introduces the marker genes. Since a variety of epitope tags and their cognate antibodies are now available commercially, one has a wide range of options from which to choose. Although the introduction of an epitope tag into the protein can in some cases change its subcellular distribution, this is relatively infrequent and usually can be overcome by changing the location of the tag within the protein. Since preparation of useful antibodies for a protein of interest is often an expensive and long-term project, the ability to detect the epitope tagged protein in vivo offers a very efficient and useful alternative. In trying to interpret CNS expressed gene function, localization of its encoded product, or correlation of its subcellular distribution in different cell types or under different conditions can provide crucial information. Obviously, the spectrum of functions one might consider is significantly different for proteins located in the nucleus than those present at the synapse! Furthermore, the redistribution of the protein in response to a stimulus can also be quite informative. For example, there are many well characterized transcriptional responses that involve regulated release of factors from cytoplasmic complexes and their entry into the nucleus in response to growth factors, cytokines, etc. (unpublished data). The ability to obtain this type of information in an efficient manner using epitope tags presents a significant advantage over the time consuming preparation of sufficiently useful antibodies to the native protein for these studies. The development of peptide tags for affinity purification is also of great utility. We have, for example, inserted the 6XHis tag into the Zipro1 locus in BAC transgenic animals for isolation of Zipro1 containing transcription complexes from cerebellar granule cells. It is now possible to utilize Ni+ chelation affinity chromatography to characterize the Zipro1 complexes using whole brain extracts from the BAC transgenic mice as has been very successfully done for His-tagged transcription factors in cultured mammalian cells. This strategy can be extended for purification of any macromolecular complex from any cell type in the brain using the BAC transgenic approach. Since the results from the animal carrying the epitope tagged protein can be directly compared to control animals, background from the purification procedure can be identified readily. While affinity purification methods are not yet fully developed for this purpose, the use of BAC transgenic animals for this purpose is a major advance over current method for identifying protein complexes that exist in vivo. When combined with the advanced mass spectrometric methods carried out in the Chait Laboratory for protein identification, this approach offers a novel and highly efficient alternative to traditional biochemical techniques. Heintz, N. (2000). """"""""Analysis of mammalian central nervous system gene expression and function using bacterial artificial chromosome-mediated transgenesis."""""""" Hum Mol Genet 9(6): 937-43. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N.""""""""A gene expression atlas of the central nervous system based on bacterial artificial chromosomes"""""""" Nature 425(2003)917-25

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000862-38
Application #
8361497
Study Section
Special Emphasis Panel (ZRG1-BCMB-Q (40))
Project Start
2011-03-01
Project End
2012-03-31
Budget Start
2011-03-01
Budget End
2012-03-31
Support Year
38
Fiscal Year
2011
Total Cost
$1,304
Indirect Cost
Name
Rockefeller University
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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