Bicistronic vector with MND promoter (Astrakhan et al., 2012 (link)) driving transcription of enhanced GFP followed by T2A sequence and 3xF. The vector had either no gene or the human SAMD9L (GenBank accession no. NM_152703 ) sequence (Origene) inserted downstream of the T2A and N-terminal 3xF using In-Fusion cloning (Takara Bio). The coding sequence ends with a stop codon and the woodchuck hepatitis virus posttranscriptional regulatory element proximal to the polyadenylation site. Site-directed mutagenesis was performed using QuikChange II (Agilent). The pCVL.SFFV.Y2 I-AniI.IRES.mCherry vector contains the spleen focus-forming virus promoter (Aubert et al., 2011 (link)). pUC19 (Takara Bio) DNA was used as the carrier DNA for the mCherry-alone condition.
Protocol detail
SAMD9L Expression via Bicistronic Vector
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Coding sequence
Gene
Human
Ires
Polyadenylation
Regulatory element
Site directed mutagenesis
Spleen focus forming virus
Stop codon
Transcription
Vector
Woodchuck hepatitis virus
Corresponding Organization :
Other organizations : University of Washington, Brotman Baty Institute, Seattle Children's Hospital, Allen Institute for Immunology
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Variable analysis
independent variables
- Presence or absence of human SAMD9L (GenBank accession no. NM_152703) sequence inserted downstream of the T2A and N-terminal 3xF using In-Fusion cloning
- Expression of enhanced GFP
- Expression of mCherry
- Bicistronic vector with MND promoter driving transcription of enhanced GFP followed by T2A sequence and 3xF
- Woodchuck hepatitis virus posttranscriptional regulatory element proximal to the polyadenylation site
- Site-directed mutagenesis performed using QuikChange II
- PCVL.SFFV.Y2 I-Ani I.IRES.mCherry vector containing the spleen focus-forming virus promoter
- PUC19 DNA used as the carrier DNA for the mCherry-alone condition
- Positive control: Bicistronic vector with MND promoter driving transcription of enhanced GFP followed by T2A sequence and 3xF, with no gene inserted downstream
- Negative control: pUC19 DNA used as the carrier DNA for the mCherry-alone condition
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