CNS gene encoding astrotactin, which supports neuronal migration along glial fibers

Science

Volumn 272, Issue 5260, 1996, Pages 417-419

  Zheng, Chen ^a   Heintz, Nathaniel ^a   Hatten, Mary E ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ASTROTACTIN; COMPLEMENTARY DNA; EPIDERMAL GROWTH FACTOR; FIBRONECTIN; IMMUNOGLOBULIN F(AB) FRAGMENT; MESSENGER RNA; NERVE PROTEIN; RECOMBINANT PROTEIN; UNCLASSIFIED DRUG;

ANIMAL TISSUE; ARTICLE; ASTROCYTE; BRAIN; CELL MIGRATION; CELL STRAIN 3T3; CENTRAL NERVOUS SYSTEM; CEREBELLUM; DNA TRANSFECTION; GENE FUNCTION; GRANULE CELL; HIPPOCAMPUS; MOUSE; NERVE CELL DIFFERENTIATION; NONHUMAN; OLFACTORY BULB; PRIORITY JOURNAL; STEM CELL;

EID: 0029947209     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5260.417     Document Type: Article

References (23)

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10. To obtain cDNAs encoding the 5′ end of the astrotactin protein, a randomly primed, P5 cerebellar granule cell cDNA library was constructed in the γ plasmid λZAPII vector. The 5′ GC14 cDNA fragment was used to screen this library. GC14 cDNAs were also isolated from a P20 mouse brain oligo(dT) λZAPII cDNA library (J Friedman, Rockefeller University)
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17. To produce recombinant GC14 protein, a cDNA fragment containing the region of one EGF and one FNIII repeat was subcloned into pGEM-3X vector (Pharmacia). Affinity purification of antibodies to GC14 was according to the protocol in Current Protocols in Molecular Biology, F M Ausubel et al., Eds. (Wiley, New York, 1994), vol. 2, suppl 27. Cell culture experiments were carried out with primary cells from cerebellums harvested on postnatal days 5 to 6 The in vitro migration culture assay was performed as described previously Fab fragments prepared from these antibodies were added at a concentration of 0.4 μg/ml. Cell images were recorded with a Panasonic optical memory disk recorder, and measurements of neuronal migration were made with Metamorph software (Universal Imaging).
18. Full-length GC14 cDNA was subcloned into pRC/ CMV vector (Invitrogen) and transfected into 3T3 cells by calcium phosphate-mediated gene transfer. Colonies resistant to G418 were screened for astrotactin expression by protein immunoblot. As a control, cell lines containing the vector alone were also tested For the cell-binding assay, a monolayer of 3T3 cells was preplated Granule and glial cells were labeled with PKH26 dye and added to the 3T3 cells. After 36 hours in vitro, the number of the labeled cells that bound and the percentage of bound cells that were differentiated were quantified by random counting of cells in 10 fields at a magnification of ×20.
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23. G Fishell and L Feng provided critical assistance with the experiments reported in Fig. 3. We gratefully acknowledge the advice of C Mason, E. Ross, and K. Zimmerman throughout these experiments, C.Z. thanks G. Dietz for many helpful discussions and for assistance in the final characterization of the astrotactin cDNAs. S. Kuhar, L. Feng, X.-L. Liu, E. Ross, and S. Vidan participated in the identification of the original set of cDNA clones used in this study; randomly primed granule cell cDNA libraries were supplied by X.-M. Qian, and D. Patterson provided expert technical assistance with in situ hybridization and photomicroscopy. Supported by NIH grant NS15429 (M.E.H ) and a McKnight Neuroscience Award (M.E.H.).