An efficient method for introducing macromolecules into living cells.
An efficient method for introducing macromolecules into living cells.
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将大分子引入活细胞的有效方法。
DOI：
10.1083/jcb.101.1.19
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发表时间：
1985-07
影响因子：
7.8
通讯作者：
WHITE, J
中科院分区：
文献类型：
--
作者：
DOXSEY, SJ;SAMBROOK, J;HELENIUS, A;WHITE, J
利用流感病毒血凝素（HA）将抗体和辣根过氧化物酶（HRP）高效、快速地批量递送到活组织培养细胞的细胞质中。利用透明质酸在细胞表面的高效表达、对红细胞的高亲和力和酸依赖的膜融合活性，开发了一种新的给药方法。该方法的独特之处在于结合和融合的介质（HA）存在于靶细胞的表面。用最近开发的永久表达HA的3T3细胞系、感染流感病毒的Madin-Darby犬肾细胞和感染携带HA基因的猴病毒40载体的CV-1细胞作为受体细胞。载蛋白的红细胞与细胞表面的HA结合，并将pH值短暂降至5.0，以触发HA的融合活性，从而进行递送。每个3T3细胞和每个CV-1细胞分别融合3 - 8个红细胞，75% -95%的细胞获得IgG或HRP。定量分析显示，每个CV-1细胞递送1.8 X 10(8)个HRP分子和1.4 X 10(7)个IgG分子，每个3T3细胞递送6.2 X 10(7)个HRP分子。通过蛋氨酸掺入蛋白质和细胞生长分裂来判断，细胞活力未受到损害。电子显微镜和荧光显微镜显示，融合的红细胞膜仍然是细胞质膜上的离散区域。该方法简便、可靠、不溶性好。同时快速地将不渗透物质输送到大量细胞中的能力，将允许对各种输送分子的命运和效果进行生化分析。
The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.
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DOI：
10.1084/jem.120.2.121
发表时间：
1964-08-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
POPE JH;ROWE WP
通讯作者：
ROWE WP
影响因子：
7.8
作者：
Mabuchi, I;Okuno, M
通讯作者：
Okuno, M
影响因子：
64.5
作者：
LIN, JJC;FERAMISCO, JR
通讯作者：
FERAMISCO, JR
影响因子：
3.2
作者：
GRAHAM, RC;KARNOVSKY, MJ
通讯作者：
KARNOVSKY, MJ
免疫球蛋白G和免疫球蛋白G片段注入HELA细胞的细胞内分布和降解。
影响因子：
7.8
作者：
McGarry, T;Hough, R;Rogers, S;Rechsteiner, M
通讯作者：
Rechsteiner, M