Microinjection preparation

C. Isolation of Oocytes for Microinjection Preparation of RNA Transport Substrates... 

Blinks, J. R., et al. (1978). Practical aspects of the use of aequorin as a calcium indicator Assay, preparation, microinjection, and interpretation of signals. Method. Enzymol. 57 292-328. 

RNA transcript combinations are prepared for injection by adding a small volume (0.5-1.0 pL) of the cRNA(s) encoding each required subunit to a 1.5 mL microfuge tube. Individual subunit cRNAs are combined and pulse spun for a few seconds and kept on ice until ready for injection. Preparation of a suitable pipette for microinjection of transcripts is imperative to ensure proper injection with minimum damage to the oocyte membrane. 

Oberholzer, Thomas, see, Fischer, Aline, 6, 37 see also Microinjection of Macromolecules in Giant Vesicles Prepared by Electroformation, 6, 285. 

It is important not to over-concentrate C3, since it tends to precipitate at high concentration (>5mg/ml). Solutions of recombinant C3 prepared in this way generally contain 3-5mg/ml, and are suitable for microinjection. Small aliquots (20 1) of the C3 transferase are stored at -20 °C. A working stock, of approximately 0.7-1 mg/ml in dialysis buffer, can be stored at 4 °C and dilutions made from this working stock at the time of microinjection. 

The concentration of each C3 protein preparation is determined with a Biorad protein assay kit, using BSA as a standard. The relative activities of C3 transferase preparations can be determined by ADP-ribosylation of recombinant Rho protein or by microinjection. For the ribosylation assay, 10 ng of recombinant RhoA protein are incubated for 1 h at 37 °C in 50 1 of ADP-ribosylation buffer (50 mM Tris-HCI, pH 7.4, 50 mM NaCI, 5 mM MgCl2, 0.3 mM GTP, 1 mM DTT, 1 iCi [ PJNAD) with varying amounts of C3 protein, ranging from 1 to 200 ng. Gel electrophoresis sample buffer is added, the samples are boiled, and proteins resolved by SDS-polyacrylamide gel electrophoresis (13.5 %). Protein is first visualized with Coomassie blue, and then the gel is destained with many washes to remove any unincorporated [ PjNAD. ADP-ribosylated rho is visualized by autoradiography. 

Each new preparation of C3 protein is also assayed by microinjection into quiescent serum-starved Swiss 3T3 cells, followed by treatment with lysophosphatidic acid, LPA (lOOng/ml for 15 or 30 min Sigma L7260) to stimulate Rho and stress fibre assembly (see Section 6.6). The lowest concentration of C3 that still blocks the LPA effect provides a measure of the relative activity of different batches of C3. 

Oberholzer, T. and Fischer, A., Microinjection of macromolecules in giant vesicles prepared by electroformation, in Giant Vesicles, P.L. Luisi and P. Walde, Eds., John Wiley Sons, New York, 2000. 

T7 RNA polymerase was encapsulated, by microinjection, inside GVs, together with DNA template and NTPSs (microinjected as well). The corresponding RNA was synthesized inside GVs prepared by the electroformation method. 

Particularly in the cases of snRNAs, tRNAs, and rRNAs, in vitro transcripts will lack covalent base and/or sugar modifications found on native cellular (RNAs (Taylor era/. 1971 Reddy and Busch, 1988 Patton, 1994 Lane etai, 1995 Maden et al., 1995). Although these modifications usually occur to in vitro transcripts over time within injected oocytes, it is sometimes desirable to study the nuclear transport of P-labeled native RNAs. For these experiments, radio-labeled RNAs are prepared by metabolic labeling of transcripts produced from endogenous genes or from genes that are microinjected, usually as plasmid DNA, into oocyte nuclei. The resultant RNAs are purified and injected into oocytes as described above. 

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