MOs (5′-CCGCCCTGGCAAGTGCGGACGA-3′) for depleting murine Cyclin B2 were synthesized by Gene Tools according to mCCNB2 (NM_007630 ; encoding Ccnb2; Gui and Homer, 2013 (link)). H2B-mCherry and MAP7-EGFP cRNAs were made from a pMDL-H2B-mCherry vector and a pGEM-MAP7-EGFP vector through in vitro transcription (T3 or T7 mMessage mMachine [Ambion] according to the manufacturer’s instructions), respectively. Mouse Ccnb2 gene (NM_007630 .2) was cloned into a pcDNA3.1-Venus vector, and its cRNA was prepared using T7 mMessage mMachine (Ambion). Mouse Ccnb1 gene (NM_172301 .3) was cloned into a pCS2+ vector, and its cRNA was prepared using SP6 mMessage mMachine (Ambion). All cRNAs were purified with RNeasy Mini kits (QIAGEN), dissolved in nuclease-free water, and stored at −80°C. A concentration of 500 ng/µl was used for microinjection. Microinjection was performed with a Nikon operating system.
Protocol detail
Murine Cyclin B2 Depletion Protocol
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Ccnb1
Ccnb2
Crnas
Gene
Microinjection
Mouse
Murine
Pgem
Transcription
Vector
Corresponding Organization : University of Chinese Academy of Sciences
Other organizations : Peking University Shenzhen Hospital, University of Hong Kong - Shenzhen Hospital, ShanghaiTech University, Model Animal Research Center, Nanjing University
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Variable analysis
independent variables
- MOs (5′-CCGCCCTGGCAAGTGCGGACGA-3′) for depleting murine Cyclin B2
- H2B-mCherry cRNA
- MAP7-EGFP cRNA
- PcDNA3.1-Venus vector containing mouse Ccnb2 gene
- PCS2+ vector containing mouse Ccnb1 gene
- Depletion of murine Cyclin B2
- Concentration of 500 ng/µl used for microinjection
- Microinjection performed with a Nikon operating system
- Positive control: Not specified
- Negative control: Not specified
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