Oocyte Microinjection

1. Thaw an aliquot of mRNA. Centrifuge 12,000g 2 min to pellet insoluble debris that can clog the injection pipet. Always handle mRNA using latex gloves.

Fig. 4. Plasmid vectors for expression of ion channels in oocytes and cultured mammalian cells. The pSP64T plasmid (18) contains an SP6 RNA polymerase promoter and the 5' and 3' UTRs from Xenopus laevis P-globin flanking a unique Bgl II restriction endonuclease site. The P-globin sequences greatly enhance translation efficiency of heterologous mRNA transcripts in oocytes. The Bgl II site is used for inserting a cDNA sequence of interest. The pRc/CMV plasmid is optimized for expression in cultured mammalian cells. Four unique restriction endonuclease sites facilitate directional insertion of a cDNA sequence adjacent to the cytomegalovirus immediate-early promoter (PCMV) and a polyadenylation signal derived from bovine growth hormone (poly-A). This plasmid also contains a neomycin resistance gene driven by the SV40 promoter for use in establishing cell lines stably expressing a cDNA. Other elements include an ampicillin resistance gene (Ampr), and bacterial origin of replication (ori, ColEl).

Fig. 4. Plasmid vectors for expression of ion channels in oocytes and cultured mammalian cells. The pSP64T plasmid (18) contains an SP6 RNA polymerase promoter and the 5' and 3' UTRs from Xenopus laevis P-globin flanking a unique Bgl II restriction endonuclease site. The P-globin sequences greatly enhance translation efficiency of heterologous mRNA transcripts in oocytes. The Bgl II site is used for inserting a cDNA sequence of interest. The pRc/CMV plasmid is optimized for expression in cultured mammalian cells. Four unique restriction endonuclease sites facilitate directional insertion of a cDNA sequence adjacent to the cytomegalovirus immediate-early promoter (PCMV) and a polyadenylation signal derived from bovine growth hormone (poly-A). This plasmid also contains a neomycin resistance gene driven by the SV40 promoter for use in establishing cell lines stably expressing a cDNA. Other elements include an ampicillin resistance gene (Ampr), and bacterial origin of replication (ori, ColEl).

2. Fabricate microinjection pipets to have tip diameter 10-15 microns. Pull glass capillaries as long as possible then break the tip with forceps that have been heated with a flame and allowed to cool (to remove trace RNA and RNase).

3. Select oocytes. Stage V and VI most typically used (see Note 3).

4. Gently inject with 20-40 nl of mRNA solution at 0.1-1.0 |g/|L (Fig. 2). Allow injection pipet to just pierce oocyte membrane and observe the oocyte "plump-up" when injected. Always change injection pipet between mRNA samples to avoid cross-contamination and plugging.

5. Incubate oocytes at room temperature or 19°C in ND-96 with antibiotics and pyruvate. Change incubation solution daily. (see Note 2).

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