von Groll Andrea, Levin Yan, Barbosa Marcia C, Ravazzolo Ana P
Departamento de Patologia Clínica Veterinaria, Faculdade de Veterinaria, and Departamento de Física Teórica, Instituto de Física, Universidade Federal do Rio Grande do Sul, Campus do Vale, Porto Alegre, RS, Brazil.
Biotechnol Prog. 2006 Jul-Aug;22(4):1220-4. doi: 10.1021/bp060029s.
A plasmid expressing the beta-galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linear DNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.
为了评估脂质体介导的环状和线性DNA转染Vero细胞的效率，使用了一种表达β-半乳糖苷酶的质粒。所得结果显示线性DNA:脂质体复合物的转染率较低。为了探究复合物的结构是否干扰转染，使用了原子力显微镜（AFM）。它证实了线性和环状凝聚物之间的差异：环状DNA:脂质体复合物呈现出约100 - 800 nm的紧密球形或圆柱形结构，而线性DNA呈现出珍珠项链状结构，珍珠大小在250至400 nm之间。基于Kuhn等人（1999年）提出的理论，使用低浓度的阳离子两亲物来中和或反转DNA电荷，以提高线性DNA的转染效率。使用这种方法，我们能够获得转基因的表达，且未观察到线性DNA脂质体递送相关的毒性。
