T3 mmessage mmachine ultra kit

For mRNA production, MPP6 and EGFP full‐length coding sequences were amplified and cloned into pBluescript II SK (+) at BamHI/EcoRV and EcoRV/XhoI restriction sites for a fusion expression of MPP6‐EGFP. The constructed plasmid was digested and linearized by PsiI, purified as a DNA template for MPP6‐EGFP mRNA transcription. T3 mMessage mMachine Ultra Kit (Ambion) was used to transcribe the initial MPP6‐EGFP mRNA, and then, Poly(A) Tailing Kit (Ambion) was used to elongate the 3′ UTR and stabilize the mRNA.
For microinjection, a thin‐wall glass tube with a built‐in guide wire (1.0 mm outer diameter, 0.8 mm inner diameter; World Precision Instruments) was pulled into two needles with Micropipette Puller P‐97 (Sutter Instrument). Then, the needle tip was bent on a Micro Forge (Narishige) at 30 degrees. MPP6‐EGFP mRNA (500‐1000 ng/µl) was loaded into the front part of the tip by guide wire‐assisted siphonage. Then, the needle was loaded onto the 3‐D electromechanical arm of a micromanipulator (Narishige) installed on a Ti‐S inverted fluorescence microscope (Nikon) and connected with a nitrogen‐driven programmable injector (Narishige). The injection time was about 10‐20 ms, and the injection volume was about 10‐20 pl.

Fidgetin and EGFP full-length coding sequences were amplified and cloned into pBluescript II SK (+) BamHI/EcoRV and EcoRV/XhoI restriction sites for fidgetin-EGFP fusion expression. The plasmid was linearized by PsiI digestion and purified as a DNA template for fidgetin-EGFP mRNA transcription. The initial fidgetin-EGFP mRNA was transcribed using the T3 mMessage mMachine Ultra Kit (Ambion, USA), followed by the Poly(A) Tailing Kit (Ambion) to extend the 3′ UTR and stabilize the mRNA.
Microinjection was performed using a micropipette Puller P-97 (Sutter Instruments, USA). The tip of the needle was bent 30 degrees using a Micro Forge (Narishige CO., Japan). MPP6-EGFP mRNA (500–1000 ng/µL) was loaded into the front of the tip by wire-assisted siphoning. The needle was then loaded onto the 3-D electromechanical arm of a micromanipulator (Narishige) mounted on a Ti-S inverted fluorescence microscope (Nikon) and connected to a nitrogen-driven programmable injector (Narishige). The injection time was approximately 10–20 milliseconds, and the injection volume was about 10–20 pL. The experiment was repeated three times.