Background: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. Methods and Results: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8 +/- 2.3 vs. 10.1 +/- 2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48 +/- 0.05 vs. 0.57 +/- 0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41 +/- 0.10 vs. 0.50 +/- 0.11; p<0.05; soleus ratio: soleus: 0.42 +/- 0.08 vs. 0.60 +/- 0.08; p<0.01) in the lower hindlimb were also observed. Conclusions: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.