This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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
| Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C et al. (2017) Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding. Anal Biochem 519:38-41 |
| Boice, Michael; Salloum, Darin; Mourcin, Frederic et al. (2016) Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells. Cell 167:405-418.e13 |
| Chait, Brian T; Cadene, Martine; Olinares, Paul Dominic et al. (2016) Revealing Higher Order Protein Structure Using Mass Spectrometry. J Am Soc Mass Spectrom 27:952-65 |
| Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Maximizing ion transmission from atmospheric pressure into the vacuum of mass spectrometers with a novel electrospray interface. J Am Soc Mass Spectrom 26:649-58 |
| Mast, Fred D; Rachubinski, Richard A; Aitchison, John D (2015) Signaling dynamics and peroxisomes. Curr Opin Cell Biol 35:131-6 |
| Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Optimizing electrospray interfaces using slowly diverging conical duct (ConDuct) electrodes. J Am Soc Mass Spectrom 26:659-67 |
| Oricchio, Elisa; Papapetrou, Eirini P; Lafaille, Fabien et al. (2014) A cell engineering strategy to enhance the safety of stem cell therapies. Cell Rep 8:1677-1685 |
| Zhong, Yu; Morris, Deanna H; Jin, Lin et al. (2014) Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels. J Biol Chem 289:26021-37 |
| Indiani, Chiara; O'Donnell, Mike (2013) A proposal: Source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 18:312-23 |
| Di Virgilio, Michela; Callen, Elsa; Yamane, Arito et al. (2013) Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching. Science 339:711-5 |
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