3d试机号开奖号合并走势图:GenCRISPR基因组编辑服务
3d试机号破解 www.7fh56.cn CRISPR-Cas9系统,被称为第三代基因编辑技术。相比于它的两位前辈ZFN系统和TALEN系统,它有着一些无可比拟的优点。首先,CRISPR-Cas9系统的可用位置更多。理论上基因组中每8个碱基就能找到一个可以用CRISPR-Cas9进行编辑的位置,可以说这一技术能对任一基因进行操作;其次,CRISPR-Cas9系统更具有可拓展性,例如可以通过对Cas9蛋白的修饰,让它不切断DNA双链,而只是切开单链,这样可以大大降低切开双链后带来的非同源末端连接造成的染色体变异风险。此外还可以将Cas9蛋白连接其它功能蛋白,实现在特定DNA序列上研究这些蛋白对细胞的影响。由于以上优点,CRISPR-Cas9被评为2013年生物学十大突破之一。
金斯瑞提供从基因信息、细胞、测序、分析到目的细胞系构建一站式技术服务,我们的GenCRISPR?基因组编辑技术,适用于任何基因的靶位点及哺乳动物细胞。金斯瑞专业的科研人员具备丰富的CRISPR基因组编辑经验,包括设计gRNA、细胞转染、单克隆细胞培养及传代、转染困难细胞系的基因编辑和构建肿瘤细胞系。服务完成后,我们将交付给您指定基因编辑后的单克隆细胞以及详细的项目报告。
请点击查看CRISPR-Cas9技术,了解更多详细信息
Case Studies: KO and KI cell lines created with GenCRISPR genome editing service
Using viral-based GenCRISPR? to develop a homozygous K-ras knock-out in HCT116 cells See more ?
- K-ras locus in human colon cancer cell line, HCT116, was knocked-out by creating indels in exon 4, as shown in the targeting strategy (Fig. 1).
- Viral encapsulation of Cas9 and gRNA delivery particles were carried out, viral titer was optimized for HCT116 cells.
- Individual clones were Sanger sequenced to select for homozygous knock-out indels of K-ras at exon 4. Sanger sequencing data (Fig. 2).
- Absence of K-ras expression in the selected clone was confirmed via Western Blot (Fig. 3).
Using GenCRISPR? to develop a glutamine synthetase (GS) knockout DG44 cell line See more ?
- A sequence optimized gRNA was designed and synthesized to target a specific region on the GS allele. DG44 cells were transfected with the construct and the cell pool was analyzed by Sanger sequencing.
- Several single clones were obtained and Sanger sequence analyzed. A single clone containing a frame shift mutation was carried forward (Fig. 1).
- The GS knockout clone was also analyzed for GS protein expression (Fig. 2).
- To assess loss of function, GS knockouts were grown in the presence or absence of increasing concentrations of glutamine (Fig. 3). GS knockouts were unable to grow in the absence of glutamine. However growth improved in the presence of increasing concentrations of glutamine, thereby indicating a functional loss in the GS knockout cell line.
- In conclusion, successful targeting of GS generated a single clone that was fully validated for lack of expression and GS function.
Using GenCRISPR? to develop a GLP-1R knock-in cell line See more ?
- AAVS1 locus in HEK 293T was used as knock-in target region. Donor DNA consisted of glucagon-like peptide 1 receptor (GLP-1R) with a puromycin selection marker, as shown in (Fig. 1).
- Cells were co-transfected with Cas9, gRNA and donor GLP-1R plasmids. Transfected cell pool was PCR analyzed and Sanger sequenced to determine whether there were GLP-1R positive clones.
- Cell pool was maintained under puromycin selection for 2 weeks and surviving cells were isolated.
- Individual clones were PCR analyzed for the Puro-GFP insert. A representative Sanger sequence of a positive clone is also shown (Fig. 2).
- GLP-1R protein expression was confirmed via immunocytochemistry staining and western blot analysis using an antibody specific to GLP-1R (Fig. 3).
Figure 1. Integration strategy for GLP-1R insertion at targeted AAVS1 site.
Figure 2. PCR analysis and Sanger sequencing of GLP-1R-positive clone.
Left: M, 1 kb DNA ladder; A, B, C, PCR amplicon from GLP-1R transfected clones. Clone A is positive for GLP-1R integration at AAVS1 site. Right: Sanger sequencing confirmation of puro-GLP-1R insertion.
Figure 3. GLP-1R expression in a representative GLP-1R clone by anti-GLP-1R antibody.
Left: Immunofluorescent staining of GLP-1R positive clones (top) compared to control (bottom) using a GLP-1R antibody. Right: Western blot analysis of a GLP-1R positive clone, compared to negative control. Anti-GLP-1R antibody detects a ~40kDa band.
Using GenCRISPR? to knock-in GFP with a puromycin selection marker See more ?
- AAVS1 locus in HEK 293T was used as knock-in target region. Donor DNA consisted of GFP and puromycin expression cassettes (Fig. 1).
- Cells were co-transfected with Cas9, gRNA and donor Puro-GFP plasmids. Control cells were co-transfected with Cas9 and Puro-GFP plasmids. GFP expression in cell pool was confirmed by fluorescence microscopy (Fig. 2).
- Cell pool was analyzed by PCR amplification using primers located outside of the homologous arm and on puromycin cassette. Amplified product was sequenced to confirm puromycin insertion (Fig. 3).
- Cell pool was treated with puromycin for 2 weeks. Single GFP-positive cells were isolated (Fig. 4).
- Individual clones were PCR analyzed for Puro-GFP insert. Clone 21 and several others were carried forward for sequence analysis (Fig. 5).
- Sanger sequencing of clone 21 shows insertion of the puromycin cassette at the targeted site (Fig. 6).
Figure 1. Targeting strategy: Puro-GFP insertion into AAVS1 locus
Adapted from; Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science (2013), 339(823);
Figure 2. GFP expression in cell pool confirmed by fluorescence microscopy
|
|
Transfection with Cas9 + gRNA + Donor Puro - GFP |
 |
Transfection with
Cas9 + Puro-GFP |
 |
Figure 3. Cell pool PCR amplification and sequencing
Figure 4. GFP-positive cells after 2 weeks of puromycin selection
Figure 5. PCR analysis of individual clones for Puro-GFP insert
Figure 6. Sequence analysis of a single clone
GenCRISPR? 服务流程图

服务说明
GenCRISPR? 细胞系构建服务 |
交付内容 |
交付时间 |
Knock-out细胞系
- 病毒(Lenti-CRISPR和AAV-CRISPR技术)或传统转染方法
- 客户指定的基因/位点区和感兴趣的细胞系*
|
- 单克隆,已确认的靶位点序列
- gRNA靶点序列设计及目标区域测序报告
|
16-20 周,最终交付时间取决于靶点的复杂程度以及细胞系生长情况 |
Knock-in细胞系
- 病毒(即将上线)或传统转染方法
- 客户指定目标基因/位点和提供的目标插入序列或突变
- 客户同样可以指定感兴趣的细胞系*
|
- 单克隆,靶序列及已确认的knock-in基因序列
- gRNA靶点序列设计及 knock-in 同源臂序列详细报告
|
20-25 周,最终交付时间取决于靶点的复杂程度、插入序列的具体情况以及细胞系生长情况 |
*一般由客户提供细胞系,如果金斯瑞提供细胞系,则需额外收费。金斯瑞除常规细胞系之外,还可以提供超过240多种的肿瘤细胞系。
|
传统转染方法(Transfection-based) |
病毒转染法(Viral-based) |
服务内容 |
- 脂质体转染(lipofection)或核转染(Nucleofaction),需提供Cas9、gRNA以及插入序列(for knock-in)
|
- 慢病毒转染(lentivirus)或腺相关病毒(adeno-associated virus),需提供Cas9、gRNA以及插入序列(for knock-in)
|
服务优势 |
- 可选择使用嘌呤霉素(puromycin)、GFP等多种标记,筛选已转染的细胞
- 较大的插入型载体,适用于敲入服务
|
- 细胞系构建速度更快
- 非分裂细胞以及重组蛋白生产常用的宿主细胞的交付率更高
- Cas9及gRNA低表达可降低脱靶效应
|
不适用于 |
- 不适用于难转染的细胞系
- 转染效率低可能会延长单克隆化的时间
|
|
适用于 |
|
|
可选服务 |
服务内容 |
附加单克隆 |
从同一个细胞库中挑选出另外一个单克隆,发给客户,附加的目标序列分析将包含在最终的报告中。 |
附加目的基因 |
客户需指定目的基因是否需同时编辑,或者哪一条靶基因需优先编辑。 |
脱靶 QC 数据 |
我们将提供给客户一份脱靶基因评估表,列举出可能会脱靶的基因,客户可根据列表选择基因进行测序,最终我们将提供给客户目标区域测序的详细报告。 |
单克隆功能验证 |
|
服务优势
- 只需客户提供目的基因,从基因合成、设计gRNA及修复模板、细胞转染、单克隆制备、到单克隆测序分析,提供真正一站式服务;
- 不存在专利问题,合法使用CRISPR-Cas9技术;
- 不使用抗生素筛选,无额外添加如GFP、或其他标签的基因(仅限基因敲除项目);
- 全球基因合成供应商,确保序列正确
;
- 体外药效学部门具备丰富细胞培养经验,已成功构建过250多种细胞系
;
- 无双抗添加细胞培养,严格确保无菌操作及无菌环境。
相关服务
询价与订购为了能够高效而准确提供报价,请首先下载并完成询价咨询表,然后填写完整的询价表通过邮件发送给我们,我们将在第一时间内给您答复。
CRISPR™产品和服务执照许可
- 金斯瑞为客户提供的GenCRISPR™产品和服务得到美国 Broad 研究所,哈佛大学和麻省理工学院授权许可。GenCRISPR™产品和服务受到US 8,697,359, US 8,771,945, US 8,795,965, US 8,865,406, US 8,871,445, US 8,889,356, US 8,889,418, US 8,895,308, US 8,906,616及多国同等专利?;?。
- GenCRISPR™产品和该服务中生成的试剂只能作为工具用于科学研发目的,不得用于以下用途:
(1)任何人体或临床的使用;(2)修改任何人类生殖体系,包括改变人类胚胎或人的生殖细胞的DNA;(3)任何作为兽药或在牲畜体内的使用;(4)制造、批发、进口、出口、运输、销售、许诺销售、市场营销及推广,或其他开发,对人类或动物的测试服务,治疗或诊断。
- GenCRISPR™服务使用者和产品的购买者:
不可转让GenCRISPR™产品和该服务中生成的试剂于第三者。GenCRISPR™产品和该服务中生成的试剂只能作为工具用于科学研发目的,不得用于任何商业目的。
客户应明确知晓上述对试剂用途的限制,并自行承担因违反该限制而产生的法律责任。
801|
204|
496|
913|
339|
239|
838|
55|
272|
822|
|