To create the MIG (MND Ires GFP)-U2AF1-Flag plasmid, U2AF1-Flag cDNA was amplified from the p3X-Flag-U2AF1 plasmid (obtained from the Kinji Ohno laboratory, Nagoya, Japan) and cloned into the MND-Ires-GFP (MIG) vector.24 (link) The S34F, S34Y, S34F/Q157R, Q157R and Q157P mutations were generated by site-directed mutagenesis (Life Technologies) using the WT MIG-U2AF1-Flag construct as a template. 293T cells were co-transfected with each MIG-U2AF1-Flag expression plasmid described above and either the GH1 or FMR1 minigene reporter constructs described previously.1 (link), 25 (link) GFP+ cells were sorted 48 hours later, followed by RNA extraction and RT-PCR as previously described.1 (link) Amplicons were visualized by polyacrylamide gel electrophoresis and quantified by densitometry (ImageJ). Three independent experiments were performed for each assay and analyzed using the Student’s t-test. Lysates were made from transfected 293T cells and immunoblotting was performed using rabbit polyclonal anti-U2AF1 antibody (Abcam) to confirm over-expression.
Protocol detail
Generating U2AF1 Mutants in MIG Vector
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293t cells
Anti antibody
Assay
Cdna
Cells
Densitometry
Ires
Mutations
Plasmid
Polyacrylamide gel electrophoresis
Rabbit
Rt pcr
Site directed mutagenesis
Student
Vector
Corresponding Organization :
Other organizations : Washington University in St. Louis, University of Rochester Medical Center, Harvard University
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Variable analysis
independent variables
- S34F mutation
- S34Y mutation
- S34F/Q157R mutation
- Q157R mutation
- Q157P mutation
- Splicing patterns of the GH1 or FMR1 minigene reporter constructs
- U2AF1 protein expression levels
- WT MIG-U2AF1-Flag construct as a template for site-directed mutagenesis
- 293T cells for co-transfection
- Positive control: WT MIG-U2AF1-Flag construct
- Negative control: Not explicitly mentioned
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