Gene transfection in primary stem-like cells of giant cell tumor of bone

Figures & data

Table 1 The quantity of DNA and reagents used and the efficiency of the chemical transfection experiments

Method	SN	DNA (μg)	Enhancer (μL)	TR (μL)	Water (μL)	CaCl2 (μL)	2 2 2 × HEPES (μL)	DMEM (mL)	Efficiency (%)
									GCT	KC
Effectene	1	0.4	3.2	8	–	–	–	2	0	0
	2	1	8	16	–	–	–	2	1	0
	3	2	16	40	–	–	–	2	–	20
	4	4	20	60	–	–	–	2	10	35
Lipofectamine	1	2	–	2	–	–	–	3	0	0
	2	4	–	4	–	–	–	3	0	0
Calcium phosphate	1	2.5	–	–	132	18.5	150	2	0	0
	2	4	–	–	215	35.5	250	2	0	0
	3	8	–	–	430	61	500	3	0	0

Note: For the Effectene transfection kit, 100 μL EC buffer was used for each reaction. DNA was plasmid pEGFPC1.

Abbreviations: SN, serial number; TR, transfection reagent.

Figure 1 Green fluorescence protein (GFP) monitored in transfected giant cell tumor of bone stromal and KC cells. Representative immunofluorescence microscopy of KC and GCT-transfected cells with eGFP vector. DAPI staining (blue) indicates the nuclei. (Oil immersion 100× magnification).

Figure 2 TWIST expression monitored in transfected giant cell tumor of bone stromal and KC cells. Representative immunofluorescence microscopy of KC and giant cell tumor of bone transfected cells with TWIST. DAPI staining (blue) indicates the nuclei. TWIST expression was detected using anti-V5 rabbit polyclonal antibody (1:250 dilutions, oil immersion 100× magnification).

Figure 3 TWIST mRNA expression. A) Real-time polymerase chain reaction of cDNA from giant cell tumor of bone stromal cells representing TWIST upregulation in transfected cells relative to TWIST untransfected cells. B) Quantitative real-time polymerase chain reaction expression of TWIST in TWIST transfected (KCT) and untransfected (KC) KC cells. Results are the average of three replicate experiments.