Journal: The Journal of Biological Chemistry
Article Title: Development of Defective and Persistent Sendai Virus Vector
Figure Lengend Snippet: Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A , the genome structure of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. B , shown is genome structure of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ), coexisting in a single cell, is shown. C and D , shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 10 5 ) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc / Klf4 / Oct4 / Sox2 as described under “Experimental Procedures.” Then 1.0 × 10 3 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days ( C ) or for the indicated days ( D ). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well ( C ) or to that of infected MEF/Nanog-GFP seeded in the well ( D ). C , shown is a comparison of reprogramming efficiency with the SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) vector and with retroviral vectors. SeVdp(M/K/O/S) , SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ); RvMX4 , coinfection of ecotropic retroviral vectors installed with c-Myc , Klf4 , Oct4 , and Sox2 separately. D , shown is a comparison of reprogramming efficiency by a single infection of SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) ( SeVdp(M/K/O/S) ) and by coinfections of SeVdp( Klf4 / Oct4 / Sox2 ) and SeVdp( Zeo / hKO / c-Myc ) ( SeVdp(K/O/S ) + SeVdp ( M )). E , characterization of mouse iPS cells generated with SeVdp( c-Myc / Klf4 / Oct4 / Sox2 ) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen ( left ), Sox2 ( middle ), and SSEA-1 ( right ), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog ( left ), expression of GFP driven by the Nanog promoter. F , gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1 , MEF; lane 2 , MEF infected with control vector (SeVdp( Bsr /Δ F / KO )); lane 3 , MEF infected with SeVdp( M/K/O/S ) on day 5 infection; lane 4 , SeVdp-iPS cell clone #2-1; lane 5 , SeVdp-iPS cell clone #9-1; lane 6 , SeVdp-iPS cell clone #13; lane 7 , SeVdp-iPS cell clone #16; lane 8 , SeVdp-iPS cell clone #21; lane 9 , mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10 , mouse ES cell (clone D3). ECAT1 , ES cell-associated transcript 1; FGF4 , fibroblast growth factor 4. G , methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic DNA was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles , unmethylated cytosine; closed circles , methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H , a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 10 3 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG) n synthesized. I , histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 10 6 SeVdp-iPS cells from clone #13 into SCID mice. a–c , low magnification; scale bar = 100 μm. d–h , high magnification observation; scale bar = 20 μm. d , cartilage; e , epidermis; f , hair follicle; g , sweat gland; h , intestinal gland. J , adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.
Article Snippet: Retroviral vectors installed separately with Oct3/4 , Sox2 , Klf4 , and c-Myc (RvMX4) were prepared as described ( ) using template DNA obtained from Addgene (Cambridge, MA).
Techniques: Infection, Cell Culture, Expressing, Microscopy, Plasmid Preparation, Generated, Incubation, Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Methylation, Sequencing, Telomerase Assay, Activity Assay, Synthesized, Derivative Assay, Injection, Mouse Assay