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. 2012 May 21;1(7):640–647. doi: 10.1242/bio.20121420

Fig. 6. In vitro synthesis of RNAs encoding CD81 and CD9 and subsequent forced expression of mRNA in oocytes.

Fig. 6.

(A) Experimental flow for in vitro synthesis of RNAs encoding mouse CD81 and CD9. (1) Subcloning of CD9 and CD81 cDNAs into plasmid vectors. The ORF corresponding to each cDNA was PCR-amplified, and the amplified DNA fragments were subcloned into the Hin dIII and Not I sites in pBluescript SKII-A85, a vector containing poly(A) repeats (comprising 85 adenines) instead of polyadenylation signal. (2) RNA synthesis. The cDNA-inserted vectors were linearized by digestion with Xho I and used as templates for RNA synthesis using the mCAP RNA Capping Kit. (B) Forced expression of mRNA encoding CD9 or CD81 in oocytes. GV-stage oocytes were collected from ovaries of CD9−/− and CD81−/− female mice and subjected to RNA injection. CD9 RNA was microinjected into CD9−/− oocytes, while CD81 RNA was injected into CD81−/− oocytes. After maturing in vitro for 24 hours, these oocytes were subjected to IVF, after which they were stained with DAPI, immunostained with anti-CD9 or anti-CD81, and observed with a confocal microscope. In each panel, scale bars: 20 µm.