Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin B, neomycin, puromycin, and Zeocin™, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin™ as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin™ is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Furthermore, Zeocin™-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin™-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin B-resistant, 47% of neomycin-resistant, and 14% of puromycin-resistant clones expressed GFP. Based on these results, we rank Zeocin™ > hygromycin B ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does indeed impact cell line development.     

稳定表达TSC1／TSC2蛋白质复合物的293T细胞系的建立

我们使用Clonetech的同源重组酶连接人TSC1、TSC2全长蛋白编码eDNA0RF到pBudCE4．1真核细胞双元表达载体上,用脂质体Lipofectamine2000介导重组质粒pBudCE4．1／TSC2／TSC1导入293T细胞,用含125μg／mLzeocin的培养基筛选稳定表达TSC1/TSC2蛋白的细胞株,并用Westemblot方法鉴定稳转细胞株的稳定性。该实验成功建立了稳定表达TSC1／TSC2蛋白的293T细胞系,从而为今后研究TSC1／TSC2蛋白的结构与功能提供实验基础。     

Induction of DNA double-strand breaks by zeocin in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response

This study aimed to test the potential of the radiomimetic chemical zeocin to induce DNA double-strand breaks (DSB) and “adaptive response” (AR) in Chlamydomonas reinhardtii strain CW15 as a model system. The AR was measured as cell survival using a micro-colony assay, and by changes in rejoining of DSB DNA. The level of induced DSB was measured by constant field gel electrophoresis based on incorporation of cells into agarose blocks before cell lysis. This avoids the risk of accidental induction of DSB during the manipulation procedures. Our results showed that zeocin could induce DSB in C. reinhardtii strain CW15 in a linear dose-response fashion up to 100 μg ml⁻¹ which marked the beginning of a plateau. The level of DSB induced by 100 μg ml⁻¹ zeocin was similar to that induced by 250 Gy of gamma-ray irradiation. It was also found that, similar to gamma rays, zeocin could induce AR measured as DSB in C. reinhardtii CW15 and this AR involved acceleration of the rate of DSB rejoining, too. To our knowledge, this is the first demonstration that zeocin could induce AR in some low eukaryotes such as C. reinhardtii.     

MAL1基因启动子区的克隆及其在大肠杆菌中的启动功能分析

目的：MAL1基因启动子区在原核生物大肠杆菌中是否有双向启动外源基因的功能。方法：利用PCR扩增MAL1基因启动子区,将不同方向的MAL1基因启动子区后接上Zeocin基因,构建成重组报告质粒,转化大肠杆菌DH5α,验证该启动子区是否双向都具有启动外源基因的功能。结果：将含不同方向启动子区的重组质粒PUCMZ1和PUCMZ2,转化大肠杆菌,转化子均有Zeoein抗性。结论：证明了MAL1结构基因上游启动子区在原核生物大肠杆菌中具有双向启动的功能,且3′-5′方向的启动能力强于5′-3′方向。