1 Q( x7 Q0 b% {- J) `/ [' ~, f5 y( AGeneration of magnetic nonviral gene transfer agents and magnetofection in vitro & T* }; N! B E) Q* bNature Protocols 2, 2391 - 2411 (2007) 0 }# a* u# |6 l; A8 \$ A; u' G
Olga Mykhaylyk, Yolanda Sánchez Antequera, Dialekti Vlaskou & Christian Plank0 L/ W. @8 R( Q! {( G
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This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force–assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3–5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.作者: 可怜的孩子 时间: 2010-4-24 20:43
转基因干细胞可向血管细胞分化。 * T2 V7 H/ Z0 R8 ^2 ] 9 @; O# r% U4 M, V2 uMIT的Daniel G. Anderson小组与Robert Langer小组研究发现： 2 P6 Q) y, b! i+ { 5 T$ a/ u0 L8 v' k! O( M5 [一种生物相容性材料（Poly(β-amino esters) (PBAE)）用于转染VEGF基因，相比于传统的脂质体转染效率更高，而且细胞毒性低。携带该基因的MSC细胞和胚胎干细胞被移植到缺血动物的后肢，发现可再生血管，同时抑制肌肉退化与组织纤维化。 & K" s* Y3 U {0 Z4 @+ H- k: E b8 a7 D& B4 M
可见开发新型用于基因治疗的载体非常必要而且很有前景。* ~4 w8 W0 z4 V+ r
- M% }% t; p3 g' yGenetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles " i6 _1 } L# fFan Yang, Seung-Woo Cho, Sun Mi Son, Said R. Bogatyrev, Deepika Singh, Jordan J. Green, Ying Mei,Sohyun Park, Suk Ho Bhang, Byung-Soo Kim, Robert Langer, and Daniel G. Andersonf % t0 v) v# B& J8 y8 e% S, D s: X4 y% ]) D; E
PNAS | February 23, 2010 | vol. 107 | no. 8 | 3317–3322作者: nanocellmatrix 时间: 2010-4-24 21:00
