The coding region of cDNA clone FI18815 was PCR amplified and cloned in frame into pENTR4 (HiFi assembly, NEB) and then into pPHW using Clonase (Life Tech.). The target sequence for GL00409 is located in the 5’UTR of Cenp-C and, therefore, this transgene is RNAi resistant. The GFP-CENP-C transgenes were constructed by Christian Lehner and contain the 5’UTR sequences upstream of the GFP sequence, and thus is RNAi sensitive. MIS12 localization was observed using a UASP regulated GFP-fusion transgene [65 (link)].
Protocol detail
Cloning and RNAi-resistant Cenp-C constructs
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5 utr
Cdna
Cenp c
Frame
Rnai
Transgene
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Variable analysis
independent variables
- Cloning of cDNA clone FI18815 into pENTR4 and pPHW vectors
- Use of target sequence for GL00409 in the 5'UTR of Cenp-C
- Construction of GFP-CENP-C transgenes containing the 5'UTR sequences upstream of the GFP sequence
- Use of UASP regulated GFP-fusion transgene for MIS12 localization
- Expression of the cloned cDNA
- Localization of MIS12 using the GFP-fusion transgene
- RNAi resistance of the Cenp-C transgene due to the use of the GL00409 target sequence in the 5'UTR
- RNAi sensitivity of the GFP-CENP-C transgenes due to the inclusion of the 5'UTR sequences upstream of the GFP sequence
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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