Research in the Macklin lab centers around the study of oligodendrocytes, which are the myelin-forming cells of the CNS. We are interested in the factors affecting them and change in their gene expression in response to stress or mutation. To this end we employ many diverse techniques from gene mapping to gene expression and from protein expression to signal transduction.
The Macklin lab maintains several mouse colonies and one rat colony carrying different mutations in the proteolipid protein (PLP) gene as well as other myelin-related genes, which reflect many human CNS disorders. Additionally the lab has generated several transgenic mice, which express reporter genes under control of the PLP promoter. The first line expresses b-galactosidase, the second expresses green fluorescent protein (GFP-S65T) and the most recent expresses enhanced GFP (EGFP) fused to the 3'untranslated region of PLP. This allows us to follow endogenous PLP expression and thus, when bred to the mutants, is invaluable for the study of oligodendrocyte development and pathology. These mice were generated by the LRI Transgenic Core Facility. The animal house in the Dept. of Neurosciences is maintained by the LRI animal facility.
An important tool in the study of oligodendrocytes and their interactions is the use of cell culture models. In our lab we use both cell lines and primary culture to investigate alterations in gene and protein expression and to analyze signal transduction pathways activated in response to growth factors. We culture primary mixed glia from mutant, wild type and transgenic animals and enrich oligodendrocytes for culture by immunopanning or shake-off methods. We also study differentiation of oligodendrocytes in slice cultures of cerebellum and cerebrum from these animals. There are 2 rooms equipped for tissue culture in the Dept. of Neurosciences providing 5 hoods and 8 double incubators.
The techniques used in the Macklin lab include routine molecular biology such as cloning of cDNAs genomic and DNA. We use Southern and Northern blotting to detect levels of DNA and RNA respectively in tissue and in cell culture. To detect the cells responsible for the expression of such RNAs, we use both radioactive and non-radioactive in situ hybridization. For low level expression of DNA and RNA, we use PCR (Polymerase Chain Reaction) and rtPCR (reverse transcription PCR). Currently we are engaged in the mapping of the gene for a novel neurological mutation within the mouse genome and are using a combination of the techniques listed as well as RFLP (Restriction Fragment Length Polymorphism). We also have access to molecular core labs, which perform basic techniques efficiently and economically. These labs include the Sequencing Core and the Gene Expression Core.
Although we have access to a cryostat and microtome in the department, for most of our studies on mice and rats, we find the Vibratome to be the instrument of choice for sectioning tissue from EGFP mice. We use antibody markers to investigate alterations in morphology or distribution of cell populations and to analyze subcellular localization of proteins. Double-label immunofluorescence allows demonstration of protein co-localization whereas DAB staining is used to analyze morphology. Within the Dept. of Neurosciences, there are 2 upright fluorescent microscopes, one inverted fluorescent microscope and a confocal laser microscope. All are connected to digital cameras for rapid image capture. We use an inverted confocal microscope to image live cell hcanged over time in brain slices from EGFP mice.
We use routine biochemistry techniques in the lab such as Western blot analysis and immunoprecipitation. We also use one- and two-dimensional gel electrophoresis, biosynthetic labeling of protein with [35S]-methionine and phosphorylation studies in order to examine the signaling pathways involved in development and survival of oligodendrocytes. Novel proteins may be isolated and sequenced by the LRI Sequencing core.
Nanthawan Avishai |
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Nan is a research technologist in the Macklin Lab.
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Qi Hao, Ph.D. |
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Qi is a research fellow in the Macklin Lab. |
Cindy Kangas |
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Cindy is a research technician in the Macklin Lab. |
Subhadra (Priya) Narayanan, Ph.D. |
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Priya is a postdoctoral fellow studying the role of neuregulin and Akt in the survival, proliferation and differentiation of oligodendrocytes in the central nervous system. Transgenic mouse models overexpressing constitutively active or dominant negative forms of Akt have been developed for these studies. |
Raymond Monk |
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Raymond is a research technologist in the Macklin Lab. |
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