Exogenous DNA expression in eukaryotic cells following microinjection.
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.
Boyd
Ann
A
Biology Department, Hood College, 401 Rosemont Ave., Frederick MD 21701, USA. boyd@hood.edu
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics*; D018014:Gene Transfer Techniques; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections / Q000379:methods* / Q000639:trends; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015203:Reproducibility of Results
D000952:Antigens, Polyomavirus Transforming; D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA