Heiser W C
Genetic Systems Division, Bio-Rad Laboratories, Hercules, California 94547.
Anal Biochem. 1994 Mar;217(2):185-96. doi: 10.1006/abio.1994.1108.
Using COS-7 and Chinese hamster ovary cells as model systems, I have examined the efficiency of gene transfer into mammalian cells by particle bombardment. The most important parameters affecting transformation efficiency are the size of the particles, the target distance, and the extent of chamber vacuum. The size of the cell culture plate also affects transformation efficiency. Factors which have little effect on transformation efficiency are the helium pressure, the gap distance, and the macrocarrier travel distance. Compared to several other gene transfer techniques, particle bombardment has the advantage of requiring a low amount of DNA and a low number of cells for successful expression, measured as either transient or stable. I also describe transformation of several murine cell lines which have not been successfully transformed, or have been transformed at only low levels using other methods. These cell lines include preadipocytes (BMS-2), macrophages (J774), and transformed pre-B cells (38B9 and 70Z/3). Compared to transformation by electroporation, lipofection, and diethylaminoethyl dextran, particle bombardment was found to give 50- to 240-fold higher levels of transient expression as measured by luciferase activity in cell extracts.
我以COS - 7细胞和中国仓鼠卵巢细胞作为模型系统,研究了通过粒子轰击将基因导入哺乳动物细胞的效率。影响转化效率的最重要参数是粒子大小、靶距离和腔室真空度。细胞培养板的大小也会影响转化效率。对转化效率影响较小的因素有氦气压力、间隙距离和宏载体移动距离。与其他几种基因转移技术相比,粒子轰击的优势在于成功表达(无论是瞬时表达还是稳定表达)所需的DNA量少且细胞数量少。我还描述了几种小鼠细胞系的转化情况,这些细胞系此前未能成功转化,或者使用其他方法仅实现了低水平转化。这些细胞系包括前脂肪细胞(BMS - 2)、巨噬细胞(J774)以及转化的前B细胞(38B9和70Z/3)。通过细胞提取物中的荧光素酶活性测定发现,与电穿孔、脂质转染和二乙氨基乙基葡聚糖介导的转化相比,粒子轰击产生的瞬时表达水平高50至240倍。