Abstract
Microinjection of nucleic acids, DNA, RNA, proteins, and any soluble material into living eukaryotic cells makes it possible to design experiments focused on single cells. In contrast facilitated transfer protocols requires hundreds of thousands of cells from which the expressed gene or intracellular effect must be detected within the culture. In addition to the immediate observable nature of the expressed product and intracellular reaction, microinjection bypasses the uptake toxicity associated with facilitated transfer of foreign material into cultured cells. The direct injection of material into the nucleus or cytoplasm allows the number of treated cells to be monitored and expression efficiencies to be observed directly. Microinjection of a hundred cells grown on small glass coverslips and subsequently counted for expression of the foreign material determines expression efficiency as a percentage of cells injected. The efficiency is based on detection of the foreign inserted gene product and does not control for relative promoter efficiency between constructs. The purpose is not to compare two constructs to each other but to monitor dual expression.
The creation of marker fluorescent proteins, such as the green fluorescent protein (GFP) in the same expression plasmid with a test gene allows the immediate observation of the GFP injected cells and within the same cells the positive or negative expression of the test gene. Expression of a foreign gene, such as SV40 T antigen cloned into an expression vector can be detected four hours after microinjection of the DNA. Fusing GFP into the same expression region of the T coding sequence labels T-GFP as a fusion protein with characteristic T immunological staining nuclear patterns but allows the cells to be studied without fixation through sequential periods of observation. The direct nature of microinjection allows comparison of gene expression in a variety of cells and the determination of the number of cells expressing the exogenous material in relationship to the number of cells injected.
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References
Diacumakos EG (1973). Methods for micromanipulation of human somatic cells in culture. Methods in Cell Biology 7: 287-311.
Capecchi MR (1980). High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22: 479-488.
Jaenisch R (1989). Transgenic animals. Science 240: 1468-1474.
Boyd AL, Samid D (1993). Review: Molecular biology of transgenic animals. J Anim Sci 71 (Suppl 3): 1-9.
Gordon JW, Ruddle FH (1985). DNA mediated genetic transformation of mouse embryos and bone marrow - a review. Gene 33: 121-136.
Woychik RP, Stewart TA, Davis LG, D’Eustachio P, Leder P (1985). An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature (London) 318: 36-40.
Pinkert CA, Widera G, Cowing C, Heber-Katz E, Palmiter RD, Flavell RA, Brinster RL (1985). Tissue specific, inducible and functional expression of the Ed MHC class II gene in transgenic mice. EMBO J 4: 2225-2230.
Boyd AL (1988). Gene transfer: A prelude to gene therapy. Methods in Cell Science 19: 231-242.
Boyd AL (1985). Expression of cloned genes microinjected into cultured mouse and human cells. Gene Anal Techn 2: 1-9.
Reeves R, Gorman C, Howard B (1985). Mini-chromosome assembly of nonintegrated plasmid DNA transfected into mammalian cells. Nucl Acids Res 13: 3599-3604.
Darnell JE (1982). Variety in the level of gene control in eukaryotic cells. Nature 297: 365-371.
Pabo CO, Sauer RT (1992). Transcription factors: structural families and principles of DNA recognition. Ann Rev Biochem 61: 1053-1095.
Johnson PT, McKnight SL (1989) Eukaryotic transcriptional regulatory proteins. Ann Rev Biochem 58: 799-839.
Ptashne M (1988). How eukaryotic transcriptional activators work. Nature 335: 683-689.
Okayama H, Berg P (1982). High-efficiency cloning of full-length cDNA. Mol Cell Biol 2: 161-170.
Murray EJ (1991). Gene transfer and expression protocols. Methods in Mol Biol, Vol. 7 Clifton, NJ: Human Press.
Southern PJ, Berg P (1982). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1: 327-341.
Subramani S, DeLuca M (1988). Applications of the firefly luciferase as a reporter gene. Genetic Engineering 10: 75-89.
Nordeen K (1988). Luciferase reporter gene vectors for analysis of promoters and enhancers. BioTechniques 6:454-456.
Chalfie M, Tu Y, Euskirchem G, Ward WW, Prasher DC (1994). Green fluorescent protein as a marker for gene expression. Science 263: 802-805.
Kain SR, Adams M, Kondepudi A, Yang TT, Ward WW, Kitts P (1995). Green fluorescent protein as a reporter of gene expression and protein localization. BioTechniques 217: 650-655.
MacGregor CR, Caskey CT (1989). Construction of plasmids that express E. coli B-galaetosidase in mammalian cells. Nucl Acids Res 17: 2365-2374.
Berg P (1981) Dissection s and reconstructions of genes and chromosomes. Science 213: 293-303.
Khoury G, Gruss P (1983) Enhancer elements. Cell 33: 313-314.
Laimins L, Khoury G, Gorman C, Howard B, Gruss P (1982). Host specific activation of transcription by tandem repeats from Simian Virus 40 and Moloney murine sarcoma virus. Proc Natl Acad Sci USA 79: 6453-6457.
Benoist C, Chambon P (1981). In vivosequence requirements of the SV40 early promoter region. Nature 290: 304-310.
Serfling E, Jasin M, Schaffner W (1985). Enhancers and eukaryotic gene transcription. Trends in Genetics 1: 224-230.
Heim R, Cubitt AB, Tsien RY (1995). Improved green fluorescence. Nature 373: 663-664.
Levy JP, Muldoon RR, Zolotukhin S, Link CJ (1996). Retroviral transfer and expression of humanized, redshifted green fluorescent protein gene into human tumor cells. Nature Biotech 14: 610-614.
White JA, Carter SG, Ozer HL, Boyd AL (1992). Cooperativity of SV40 T antigen and ras in progressive stages of transformation of human fibroblasts. Exp Cell Res 203: 157-163.
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Boyd, A. Exogenous DNA expression in eukaryotic cells following microinjection. Methods Cell Sci 24, 115–122 (2002). https://doi.org/10.1023/A:1024401924835
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DOI: https://doi.org/10.1023/A:1024401924835