Abstract
Mammalian-cell microinjection is a powerful method for analyzing the in vivo functions of viral genes and viral gene products. By microinjection, a controlled amount (ranging from 1 to many thousands of copies) of a viral or cellular gene, a protein product of a gene, a polypeptide fragment encoding a specific protein domain, or an RNA molecule can be delivered into a target cell and the functional consequences analyzed. Injection of DNA into the nuclei of cultured mammalian cells provides a sensitive bioassay for gene expression. The product of a single injected gene copy can be detected using standard immunofluorescent, immunochemical, and autoradiographic techniques (1). The direct analysis of protein function by microinjection has been facilitated by the ability to produce biologically active recombinant proteins and protein fragments encoded by many interesting genes.
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References
Capecci, M. (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells Cell 22, 479ā488
Shenk, T., (1996) Adenoviridae: the viruses and their replication, in Virology (Fields, B. et al., eds.), Lippincott-Raven, New York, pp. 2111ā2148.
Lillie, J. W., Loewenstein, P. M., Green, M. R., and Green, M., (1987) An adenovirus El A protein region required for transformation and transcriptional repression Cell 46, 1043ā1051.
Green, M., Loewenstein, P. M., Pusztai, R., and Symington, J.S., (1988) An adenovirus El A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis Cell 53, 921ā926.
Song, C-Z., Tierney, C. J., Loewenstein, P. M., Pusztai, R., Symington, J. S., Tang, Q.-Q., Toth, K., Nishkawa, A., Bayley, S. T., and Green, M., (1995) Transcriptional repression by human adenovirus E1A N-terminus/conserved domain 1 polypeptides in vivo and in vitro in the absence of protein synthesis. J Biol Chem 270, 23,263ā23,267.
Stabel, S., Argos, P., and Phihpson, L. (1985) The release of growth arrest bymicroinjection of adenovirus. EMBO J 4, 2329ā2336
Kaczmarek, L., Ferguson, B., Rosenberg, M., and Baserga, R., (1986) Induction ofcellular DNA synthesis by purified adenovirus El A proteins.Virology 152, 1ā10.
Ruley, H. E., (1983) Adenovirus early region 1A enables viral and cellulartransforming genes to transform primary cells in culture. Nature (London)304, 602ā606
Mulcahy, L. S., Smith, M. R., and Stacey, D. W., (1985) Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature (London) 313, 241ā243
Dobrowolski, S., Harter, M., and Stacey, D. W., (1994) Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3cells. Mol. Cell. Biol. 14, 5441ā5449
Riabowal, K. T., Vosatka, R. J., Ziff, E. B., Lamb, N. J., and Feramisco, J. R., (1988) Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells. Mol. Cell. Biol. 8, 1670ā1676
Kovary, K., and Bravo, R., (1992) Thejun and fos protein families are both requiredfor cell cycle progression in fibroblasts. Mol Cell. Biol 11, 4466ā4472.
Anas, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Kann, M., Feramisco, J., and Montminy, M., (1994) Activation of cAMP and mitogen responsive genes relies on a common nuclear factor Nature (London)370, 226ā229.
Lawrence, J. B., and Singer, R. H., (1986) Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45, 407ā415.
Kislauskis, E. H., Li, Z., Singer, R. H., and Taneja, K.L. (1993) Isoform-specific 3ā-untranslated sequences sort Ī±-cardiac and Ī²-cytoplasmic actin messengerRNAs to different cytoplasmic compartments. J Cell Biol 123, 165ā172.
Nonradioactive in situ hybridization application manual, 2nd ed Boehringer Mannheim, Mannheim, Germany.
Harlow, E., and Lane, D., (1988) Antibodies A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
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Ā© 1999 Humana Press Inc., Totowa, NJ
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Green, M., Thorburn, A., Loewenstein, P.M. (1999). Cell Microinjection. In: Wold, W.S.M. (eds) Adenovirus Methods and Protocols. Methods in Molecular Medicineā¢, vol 21. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-551-4:169
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DOI: https://doi.org/10.1385/0-89603-551-4:169
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-551-5
Online ISBN: 978-1-59259-603-4
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