Abstract
Capillary microinjection is one of the most efficient methods for introducing macromolecules into living mammalian cells (Proctor 1992). Samples can be delivered directly into either the nucleus or the cytoplasm. In synchronized cell cultures, the molecules can be transferred at well defined stages of the cell cycle and modifications of culture conditions are possible, before, during, or after injection. Therefore, microinjection of target specific inhibitors (e.g., antibodies, synthetic peptides, antisense mRNAs) or activators (e.g., proteins, peptides, cDNAs under the control of a constitutive promoter) has been a useful tool to study the in vivo function of proteins in cell proliferation and cell cycle control.
Keywords
- Electroporation Efficiency
- Fluorescein Dextran
- Microinjection Experiment
- Automate Injection System
- Chase Medium
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Ansorge W, Pepperkok R (1988) Performance of an automated system for capillary micro-injection into living cells. J Biochem Biophys Meth 16:283–292
Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G (1993) Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 7:812–821
Chang DC, Chassy B, Saunders JA, Jordon CA (eds) (1992) Handbook of electroporation and electrofusion. Academic Press, San Diego
DeWet JR, Wood K, DeLuca M, Helsinki D, Subramini S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737
Gautier-Rouviere C, Fernandez A, Lamb NJC (1990) Ras-induced c-fos expression and proliferation in living rat fibroblasts involves C-kinase activation and the serum response element pathway. EMBO J 9:171–180
Lane HA, Fernandez A, Lamb NJC, Thomas G (1993) p70sk6 function is essential for G1 progression. Nature 363:170–172
Lorenz P, Pepperkok R, Ansorge W, Pyerin W (1993) Cell biological studies with monoclonal and polyclonal antibodies against human casein kinase II subunit ß demonstrate participation of the kinase in mitogenic stimulation. J Biol Chem 268:2733–2739
Lukas J, Pagano M, Staskova Z, Draetta G, Bartek J. (1994) Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumor cell lines. Oncogene 9: 707–718
Mittman S, Flaming DG, Copenhagen DR, Belgum JH (1987). Buble pressure measurement of micropipette tip outer diameter.. Neurosci Meth 22:161–166
Pagano M, Pepperkok R, Lukas J, Baldin V, Ansorge W, Bartek J, Draetta, G (1993) Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts. J Cell Biol 121 101–111
Pepperkok R, Zanetti M, King R, Delia D, Ansorge W, Philipson L, Schneider C (1988) Automatic microinjection system facilitates detection of growth inhibitory mRNA. Proc Natl Acad Sci USA 85:6748–6752
Pepperkok R, Scheel J, Horstman H, Hauri HP, Griffiths G, Kreis TE (1993a). ß-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell 74:71–82
Pepperkok R, Herr S, Lorenz P, Pyerin W, Ansorge W (1993b) System for quantitation of gene expression in single cells by computerized micro-imaging: application to c-fos expression after microinjection of anti-casein kinase II (CKII) antibody. Exp Cell Res 204:278–285
Pepperkok R, Lorenz P, Ansorge W, Pyerin W (1994) Casein kinase II is required for G0/G1, early G1 and G1/S phase of cell cycle. J. Biol. Chem. 269:6986–6991
Potter H (1993). Application of electroporation in recombinant DNA technology. Methods Enzymol 217:461–478
Proctor GN (1992) Microinjection of DNA into mammalian cells in culture: theory and practise. Meth Mol Cell Biol 3:209–231
Sorrentino V, Pepperkok R, Davis RL, Ansorge W, Philipson L (1990) Cell proliferation inhibited by MyoD1 independently of myogenic differentiation. Nature 345:813–815
Tam SW, Theodoras AM, Shay JW, Draetta GF, Pagano M (1994) Differential expression and regulation of cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for cyclin D1 function in G1 progression. Oncogene 9:2663–2674
Todorov IT, Pepperkok R, Philipova RN, Kearsy SE, Ansorge W, Werner, D (1994) A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division. J Cell Sci 107:253–265
Twamley-Stein GM, Pepperkok R, Ansorge W, Courtneidge SA (1993) The src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH3T3 cells. Proc Natl Acad Sci USA 90:7696–7700
Weaver JC (1993) A general phenomenon for manipulating cells and tissue. J Cell Biochem 51:426–435
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Pepperkok, R. (1996). Microinjection and Electroporation of Macromolecules into Live Cells. In: Pagano, M. (eds) Cell Cycle — Materials and Methods. Springer Lab Manual. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-57783-3_7
Download citation
DOI: https://doi.org/10.1007/978-3-642-57783-3_7
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-58066-9
Online ISBN: 978-3-642-57783-3
eBook Packages: Springer Book Archive