pAcGFP1-Mem載體描述 pAcGFP1-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43 (1), and the Aequorea coerulescens green fluorescent protein AcGFP1. (Excitation maximum = 475 nm; emission maximum = 505 nm.) The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (2). The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets AcGFP1 to cellular membranes, and the plasma membrane in particular. Expression of AcGFP1-Mem is driven by the immediate early promoter of CMV (PCMV IE). The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
pAcGFP1-Mem can be transfected into mammalian cells using any standard method. If required, stable transformants can be selected using G418 (3). Expression of AcGFP1-Mem in mammalian cells results in strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This membrane labeling also permits the study of fine cellular processes such as neuronal axons (4), leading edges of migrating cells, filopodia, or microvilli on cell surfaces. pAc- GFP1-Mem cannot be used as an exclusive plasma membrane marker because it also partially labels intracellular membranes.
Propagation in E. coli
Suitable host strains: DH5α, HB101, and other general-purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
References
1. Skene, J. H. P. & Virag, I. (1989) J. Cell. Biol. 108:613–625.
2. Haas, J., et al. (1996) Curr. Biol. 6:315–324.
3. Gorman, C. (1985) In DNA cloning: a practical approach, vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
4. Moriyoshi, K., et al. (1996) Neuron 16:255–260.
