遗传霉素 (geneticin, G418)是一种氨基糖苷类抗生素, 是最常用的细胞稳定转染抗性筛选试剂。G418能阻断蛋白质合成，对细菌、酵母、高等植物以及哺乳动物细胞均具有氨基糖苷类毒性，用于真核细胞从转座子Tn5和Tn601稳定转染新霉素 (neo)抗性基因的选择和维持，经常用于基因转移、基因敲除、抗性筛选以及转基因动物等方面。G418硫酸盐是G418的硫酸盐形式，其cas号是108321-42-2。
G418 Solution经过滤除菌，一般工作浓度为50-1000 μg/ml不等，不同类型的细胞需要根据实验确定最佳浓度，在具有相对稳定基因组的细胞 (如CHO细胞)中，一旦筛选出稳定细胞株后，无需加入G418即可继续培养。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
操作步骤 (仅供参考)：
1、由于每种细胞对G418的敏感性不同，而且不同批次G418的活性也不尽相同，所以在筛选之前，需要确定G418的最佳筛选浓度，一般在50-1000 μg/ml范围内进行筛选。
2、将细胞制备成细胞悬液，并稀释到1000个/ml。
3、加入不同量的G418 Solution (50 mg/ml)，选择在10~14天内使细胞全部死亡的最低G418浓度来进行下一步的筛选试验。
注意事项：
1、尽量减少反复冻融的次数，以免失效。
2、基因转染到细胞内后，需要一段时间才能表达出蛋白质，所以筛选不宜太早。
3、筛选也不宜太迟，一般要在转染24h之后才开始加G418筛选，因为转染了外源基因的细胞代谢负荷较大，增值较慢，时间长了就会被没有外源基因转入的细胞所淹没，最终导致筛选不出阳性克隆。
4、随着细胞代谢，G418的浓度和活性都会下降，应每3-5天更换一次含有G418的筛选液，这时药物浓度可以降至200 μg/ml。
5、加药筛选约5-7天左右，细胞会大量死亡，为了减少死亡细胞对阳性克隆的不利影响以及增加阳性克隆的获得率，可以应用套环法或刮除法结合有限稀释法来筛选阳性克隆，加药后在高倍镜下，刮除阴性克隆，消化阳性克隆后继续筛选培养或者用套环套住阳性克隆，在套环内加胰蛋白酶消化，把消化液吸到另外一个新的孔中培养，最后再用有限稀释法把阳性克隆在96孔板中筛选。
6、一般经过4周左右的筛选，得到的阳性克隆都比较稳定，但是外源基因如果没有整合到基因组中的话，目的基因还是很容易丢失的，再次筛选往往是必不可少的，经过2次以上的筛选之后才能找到遗传稳定的细胞克隆。
7、注意无菌操作，尽量避免污染。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Geneticin (G418) is an aminoglycoside antibiotic, and it is the most commonly used reagent for resistance screening in stable cell transfection. G418 can block protein synthesis and exhibits aminoglycoside toxicity against bacteria, yeast, higher plants and mammalian cells. It is applied to the selection and maintenance of eukaryotic cells stably transfected with the neomycin (neo) resistance gene from transposons Tn5 and Tn601, and is frequently used in gene transfer, gene knockout, resistance screening and transgenic animal research. Geneticin sulfate is the sulfate form of G418, with a CAS number of 108321-42-2.
G418 Solution is sterilized by filtration, with a typical working concentration ranging from 50 to 1000 μg/ml. The optimal concentration should be determined experimentally according to different cell types. For cells with a relatively stable genome (e.g., CHO cells), continuous culture can be conducted without adding G418 once stable cell lines are obtained. This reagent is for research use only and is not suitable for clinical diagnosis or any other purposes.
Operating Procedures (for reference only)
1. Since the sensitivity of each cell type to G418 varies, and the activity of different batches of G418 is not identical, it is necessary to determine the optimal screening concentration of G418 before screening, which is generally in the range of 50-1000 μg/ml.
2. Prepare the cells into a single-cell suspension and dilute it to a density of 1000 cells/ml.
3. Add different volumes of G418 Solution (50 mg/ml). Select the minimum G418 concentration that causes complete cell death within 10-14 days for subsequent screening experiments.
Precautions
1. Minimize the number of freeze-thaw cycles to prevent loss of activity.
2. A certain period of time is required for the expression of target proteins after gene transfection into cells, so screening should not be performed too early.
3. Screening should not be delayed excessively either. Generally, G418 should be added for screening 24 hours after transfection. This is because cells transfected with exogenous genes bear a heavier metabolic burden and proliferate more slowly. If the screening is delayed, these transfected cells will be outcompeted by non-transfected cells, ultimately resulting in failure to isolate positive clones.
4. The concentration and activity of G418 will decrease with cellular metabolism. The screening medium containing G418 should be replaced every 3-5 days, and the drug concentration can be reduced to 200 μg/ml at this time.
5. A large number of cells will die approximately 5-7 days after drug addition. To reduce the adverse effects of dead cells on positive clones and improve the yield of positive clones, the ring cloning method or scraping method combined with the limiting dilution method can be used for screening. After drug addition, scrape off negative clones under a high-power microscope, digest the positive clones and continue screening culture; alternatively, encircle positive clones with cloning rings, add trypsin inside the rings for digestion, aspirate the digested solution into new wells for culture, and finally screen positive clones in 96-well plates using the limiting dilution method.
6. Generally, positive clones obtained after about 4 weeks of screening are relatively stable. However, if the exogenous gene is not integrated into the genome, the target gene is still prone to loss. Re-screening is often essential, and genetically stable cell clones can only be obtained after more than two rounds of screening.
7. Pay attention to aseptic technique to avoid contamination as much as possible.
8. For your safety and health, wear a lab coat and disposable gloves during operation.
ZJ26F0333245
危险品化学品经营许可证（带存储）