EasySep™小鼠TIL（CD45）正选试剂盒
产品号 #73342_C
抗生素；蛋白质合成抑制剂
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总览
嘌呤霉素是一种氨基核苷类抗生素，源自白黑链霉菌（Streptomyces alboniger），可作为蛋白质合成抑制剂。它与靶核糖体A位点结合，并转移到正在生长的多肽链上，导致多肽链提前终止（Lührmann等；Rodriguez-Fonseca等；Azzam和Algranati）。嘌呤霉素被嘌呤霉素-N-乙酰转移酶N-乙酰化后，会形成一种生物无活性的形式，这使得该基因可用作选择性抗性标记（Vara等，1985）。
细胞系开发
·筛选表达嘌呤霉素-N-乙酰转移酶抗性基因的细胞作为研究工具（de la Luna和Ortín；Iwaki等）。
·可用于CRISPR/Cas9哺乳动物基因编辑，通过筛选成功敲入Cas9基因的嘌呤霉素抗性基因（Park等）。
癌症研究
·经多种细胞系测试，显示其具有抗肿瘤活性（Foley和Eagle）。
细胞类型
癌细胞及细胞系，CHO细胞，杂交瘤细胞，多能干细胞
种属
人，小鼠，非人灵长类，其它细胞系，大鼠
应用
抗生素
研究领域
癌症，细胞系制备，杂交瘤制备
CAS 编号
58-58-2
化学式
C₂₂H₂₉N₇O₅ · 2HCl
纯度
≥98%
产品说明书及文档
请在《产品说明书》中查找相关支持信息和使用说明，或浏览下方更多实验方案。
应用领域
本产品专为以下研究领域设计，适用于工作流程中的高亮阶段。探索这些工作流程，了解更多我们为各研究领域提供的其他配套产品。
相关材料与文献
技术资料 (1)
文献 (10)
CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function. PloS one 2014
Abstract
Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.
Targeting of Herpes Simplex Virus 1 Thymidine Kinase Gene Sequences into the OCT4 Locus of Human Induced Pluripotent Stem Cells PLoS ONE 2013 NOV
Abstract
The in vitro differentiation of human induced pluripotent stem cells (hiPSC) to generate specific types of cells is inefficient, and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC, we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK) suicide gene at the endogenous OCT4 (POU5F1) locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only, we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV) prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus, we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN) and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences, flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed, three contained the HSV1-TK transgene at the OCT4 locus, but they were not sensitive to GCV. The other six clones were GCV-sensitive, but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days, indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed.
Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells PLoS ONE 2012 APR
Abstract
The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular, in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies.
更多信息
| 种属 | Human, Mouse, Non-Human Primate, Other, Rat |
|---|---|
| Cas Number | 58-58-2 |
| Chemical Formula | C₂₂H₂₉N₇O₅ · 2HCl |
| 纯度 | ≥ 98% |
