We constructed a synthetic humanized SF3B1 ORF (mxSF3B1) by ligating the N-terminal half of the mouse SF3B1 ORF and the C-terminal half of the Xenopus SF3B1 ORF, followed by replacement of three amino acid residues in the Xenopus ORF with three human-type residues. This was carried out because it was reported previously that cloning of human SF3B1 cDNA is impossible  and also because we have experienced similar difficulties with mouse SF3B1 cDNA. The mouse SF3B1 ORF was amplified from mouse brain Quick-Clone cDNA (Clontech, Mountain View, CA, USA) using FastStart HiFi DNA polymerase (Roche Diagnostics, Mannheim, Germany) and a primer pair of T013-FB and T021-RH. A mouse SF3B1 ORF of approximately 4 kb was successfully amplified, although it was difficult to clone into vectors as a result of its toxicity in Escherichia coli. Therefore, the N-terminal half of mouse SF3B1, a fragment of approximately 1.5 kb obtained by BamHI-XhoI digestion, was cloned into a pBluescript II SK(+) vector (Stratagene, La Jolla, CA, USA). The C-terminal half of the gene for Xenopus SF3B1 was cloned by PCR using the Xenopus laevis embryo LAMBDA cDNA library (Stratagene) and a primer pair of xenoSAP155-F1/Bm and xenoSAP155-R1 (Table 1). The PCR product was cloned into a pBluescript II KS(+) vector (Stratagene) using BamHI and HindIII restriction sites. The plasmid, designated pCMVHA-mxSF3B1, was constructed by insertion of DNA fragments into the pcDNA3.1(−) vector (Invitrogen): the HA-Nhe-Bam-adaptor fragment (5′-GCTAGCACCATGAGAGGTTCGAACTACCCCTACGACGTGCCAGACTACGCTTCCCTGGGATCACTCGAGGAATTCGTCGACGGTACCGGGCCCGGATCC-3′) containing an initiation codon followed by the HA-epitope (YPYDVPDYA); the N-half of mouse SF3B1 (NheI-ScaI) fragment; and the C-half of the Xenopus SF3B1 (ScaI-HindIII) fragment. The resulting plasmid was used as a template for the site-directed mutagenesis to convert the Xenopus-unique amino acids. Three amino acids in the Xenopus SF3B1 (i.e. Val1247, Glu1291 and Thr1303) were changed to the human-type Leu, Asp and Ile, respectively, using the PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Shiga, Japan). The primers used were: V1247L forward, V1247L reverse, E1291D forward, E1291D reverse, T1303I forward and T1303I reverse (Table 1). To generate the mutant allele, R1074H (single amino acid replacement of Arg1074 with a His), we performed PCR-based mutagenesis using the primers xSAP155-mutant-F and xSAP155-mutant-R (Table 1). Finally, the HA-mxSF3B1 ORFs (wild-type and R1074H) were inserted into a modified pCLXSN retroviral vector (IMGENEX, San Diego, CA, USA), in which the SV40 promoter-neo(r) sequence had been replaced with the IRES-hyg (r) from the pIREShyg vector (Clontech), resulting in pCLXIH-SF3B1(Wt) and pCLXIH-SF3B1(R1074H).
The target sequences for SF3B1 knockdown were shSF3B1 (3989) 5′-GCACAGCTACTTCACACCTTA-3′ and shSF3B1 (4145) 5′-GCCAGTAGTGACCAAGAACAC-3′. These sequences were inserted into the pENTR/U6 vector (Invitrogen) in accordance with the manufacturer’s instructions. The resulting vectors, designated pENTR/shSF3B1 (3989) and pENTR/shSF3B1 (4145), had SF3B1 shRNA sequences under the control of the U6 promoter. The knockdown platform vector was constructed by ligating the Gateway Vector Conversion System Reading Frame Cassette C.1 (Invitrogen) and the cytomegalovirus promoter-EGFP fragment from pEGFP-N2 vector (Clontech) into the BamHI-KpnI restriction sites of the pLenti6/V5-GW/lacZ vector (Invitrogen). pLenti/shSF3B1 (3989) and pLenti/shSF3B1 (4145) vectors were constructed by recombination of the attL cassettes from the pENTR/shSF3B1 vectors into the knockdown platform vector using the LR Clonase II enzyme mix (Invitrogen).
For viral production, 293-EBNA cells (Invitrogen) were transfected with the packaging mix and the above viral vector using the TransIT-LT1 reagent (Mirus, Madison, WI, USA). Medium was exchanged 8 h after transfection, and the culture supernatant was collected after culturing for another 2 days. The collected culture supernatant was filtered through a 0.45 μm syringe filter (Millipore, Billerica, MA, USA) and used as the virus solution. The virus solution for SF3B1 expression was concentrated by ultracentrifugation at 50 000 g for 100 min at 4 °C. WiDr cells were transduced with the virus solution in the presence of 6 μg·mL−1 Polybrene (Sigma-Aldrich, St Louis, MO, USA).