Journal: Cell Journal (Yakhteh)

Article Title: Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

doi:

Figure Lengend Snippet: Physical map of the vectors and electroporation procedure. A. Both pVASA -EGFP and pOLIG2 -EGFP benefit from dual selection system including neomycin and puromycin. No difference was seen in transformation efficiency despite the different vectors’ sizes and B. Human embryonic stem cells (hESCs) were seeded onto 50 cm plates and grown in appropriate medium until they reached 70-80% confluency. Following incubation in Tryp/LE at 37˚C for 5 minutes the cells were singled. For electroporation, the cells were counted and resuspended in phosphate buffer saline (PBS) at a concentration of 1×10 6 , after which 700 μl of cell suspension was mixed with 20-60 μg of linear plasmid DNA in a sterile electroporation cuvette. The voltage varied from 240 to 300 V. Immediately after electroporation, the cells were removed from the cuvette and plated on three 10 cm diameter tissue culture dishes in complete medium. After 48 hours, the plates were washed twice with phosphate buffer saline (PBS), then replenished with complete medium. MEF; Mouse embryonic fibroblast.

Article Snippet: In brief, 600 μl of the previously singled cells that contained 10-60 μg linearized plasmid was transferred into the electroporation cuvette (BioRad, USA, #165-2088).

Techniques: Electroporation, Selection, Transformation Assay, Incubation, Saline, Concentration Assay, Suspension, Plasmid Preparation, Sterility

Journal: Cell Journal (Yakhteh)

Article Title: Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

doi:

Figure Lengend Snippet: In vitro transfection of H5, H6 and human foreskin fibroblast (hFF) cell lines by lipofectamine and electroporation. A. A comparison of chemical and physical technique efficiencies for gene transformation confirmed a higher rate of transfection for electroporation in individual cell lines. The graph shows the averages of three independent experiments. Error bars represent the standard deviation. **; P<0.01 and B. Flow cytometric analysis of EGFP expression in three independent experiments. After 48 hours of gene delivery, we analyzed transient expression of EGFP by flow cytometry. hFF cells showed the highest percentage (27%) of expression when compared with the other cells. Interestingly, a comparison of the two different human embryonic stem cells (hESCs) demonstrated that Royan H5 exhibited greater transformation potential (approximately 17 vs. 10%).

Article Snippet: In brief, 600 μl of the previously singled cells that contained 10-60 μg linearized plasmid was transferred into the electroporation cuvette (BioRad, USA, #165-2088).

Techniques: In Vitro, Transfection, Electroporation, Comparison, Transformation Assay, Standard Deviation, Expressing, Flow Cytometry

Journal: Cell Journal (Yakhteh)

Article Title: Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

doi:

Figure Lengend Snippet: Transient expression of EGFP in Royan H6 and human foreskin fibroblast (hFF) cell lines. A, D. Bright-field images of human embryonic stem cells (hESCs) and hFF cells, B, E. Fluorescent images and C, F. Merged bright-field and fluorescent images. The pictures showed that hFF responded to electroporation more efficiently than hESCs.

Article Snippet: In brief, 600 μl of the previously singled cells that contained 10-60 μg linearized plasmid was transferred into the electroporation cuvette (BioRad, USA, #165-2088).

Techniques: Expressing, Electroporation

Journal: Nature

Article Title: Peripheral deletion of self-reactive B cells

doi: 10.1038/354308a0

Figure Lengend Snippet: RNA analysis of transgenic and non-transgenic mice. a, Northern blot analysis shows reduction in transgenic κ-light chain expression in spleen and lymph nodes of Dbl-Tg mice. After hybridization with Vκ 3-83 probe4 and decay of the radioactive signal, the filter was hybridized with the MHC class 1-specific pll2a probe28. b, Polymerase chain reaction (PCR) detection of Vκ 3-83 RNA in the livers of peripherally deleting mice. Lanes 1, 3, 5 and 7, liver; lanes 2, 4, 6 and 8, bone marrow. Lanes 1 and 2, mouse 2080 (Dbl-Tg); lanes 3 and 4, mouse 2081 (Dbl-Tg); lanes 5 and 6, mouse 2082 (Ig–Tg); lanes 7 and 8, Non-Tg littermate. Complementary DNA was synthesized and amplified with 15 cycles of PCR, run on a 1.2% agarose gel, transferred to a Zetaprobe membrane (BioRad) and hybridized with probes recognizing gene transcripts of 3-83 Vκ or the GTP-binding protein Gαs (ref. 29). Lane M, marker DNA fragments of 587, 434 and 267 bp.

Article Snippet: Complementary DNA was synthesized and amplified with 15 cycles of PCR, run on a 1.2% agarose gel, transferred to a Zetaprobe membrane (BioRad) and hybridized with probes recognizing gene transcripts of 3-83 V κ or the GTP-binding protein G α s (ref. 29 ).

Techniques: Transgenic Assay, Northern Blot, Expressing, Hybridization, Polymerase Chain Reaction, Synthesized, Amplification, Agarose Gel Electrophoresis, Membrane, Binding Assay, Marker