pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP BioVector? 質粒 / pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP BioVector? Plasmid

數(shù)據(jù)表 / Datasheet

質粒信息

• 名稱：BioVector? pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP 質粒

• 詳細名稱：含 UCOE 穩(wěn)定表達元件、SFFV 啟動子驅動的 Zim3-dCas9 融合蛋白及 P2A-BFP 報告基因的慢病毒載體

• 載體類型：慢病毒表達載體（第三代 HIV-1 來源）

• 載體骨架：pHR（含 LTR、包裝信號、RRE、cPPT/CTS 等元件）

• 宿主菌：推薦使用 BioVector? Stbl3 或 NEB? Stable 感受態(tài)細胞，避免重組

• 抗性：氨芐青霉素（Ampicillin，100 μg/mL）

• 拷貝數(shù)：低拷貝

• 質粒大小：約 12–14 kb（具體長度以測序為準）

載體元件

培養(yǎng)與保存

• 質粒提取：使用 BioVector? 無內毒素質粒大提試劑盒；建議在含氨芐青霉素的 BioVector? LB 培養(yǎng)基中擴增，30°C 培養(yǎng)以減少重組

• 保存條件：短期 -20°C，長期 -80°C 保存甘油菌或純化質粒

包裝與轉導

• 包裝細胞：推薦使用 BioVector? HEK293T 細胞

• 包裝質粒：需配合第二代或第三代包裝輔助質粒（如 BioVector? psPAX2 和 BioVector? pMD2.G）

• 轉染試劑：BioVector? 聚乙烯亞胺（PEI）或 BioVector? Lipofectamine? 3000

• 病毒收獲：轉染后 48–72 小時收集上清，0.45 μm 濾膜過濾，可濃縮后使用

• 宿主細胞：廣泛適用于分裂與非分裂細胞，包括 BioVector? NHEK、BioVector? HEK293、BioVector? Jurkat 等

特征與用途

• CRISPRi 高效抑制：Zim3 是新一代 KRAB 結構域，與 dCas9 融合后可實現(xiàn)對靶基因轉錄的強效、特異性抑制，脫靶效應低，尤其適合基因功能喪失性研究

• 長期穩(wěn)定表達：UCOE 元件可抵抗位置效應和啟動子沉默，確保在干細胞、原代細胞及體內模型中持續(xù)穩(wěn)定的 CRISPRi 活性

• 報告系統(tǒng)：BFP 熒光報告基因可實時監(jiān)測病毒轉導效率及表達水平，便于分選（流式細胞術）和單克隆篩選

• 單載體系統(tǒng)：所有元件整合于單個慢病毒載體，簡化了病毒制備和細胞工程流程

• 應用方向：

  • 基因功能篩選（CRISPRi 文庫）

  • 信號通路中關鍵基因的失活研究

  • 原代細胞（如 T 細胞、神經(jīng)細胞、角質形成細胞）的基因調控

  • 體內遞送及基因治療模型構建

注意事項

• 該質粒為第三代慢病毒載體，SIN 設計提升了安全性，但仍應在生物安全二級（BSL-2）條件下操作

• 因含有長末端重復序列和重復元件，建議在 30°C 下培養(yǎng)細菌，避免重組缺失

• 使用前應通過測序或酶切驗證完整性

Plasmid Information

• Name: BioVector? pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP Plasmid

• Detailed Name: Lentiviral Vector Containing UCOE Stabilizing Element, SFFV Promoter-Driven Zim3-dCas9 Fusion Protein, and P2A-BFP Reporter

• Vector Type: Lentiviral expression vector (third-generation HIV-1 derived)

• Backbone: pHR (containing LTR, packaging signal, RRE, cPPT/CTS, etc.)

• Host Strain: BioVector? Stbl3 or NEB? Stable competent cells recommended to avoid recombination

• Selection: Ampicillin (100 μg/mL)

• Copy Number: Low copy

• Plasmid Size: Approximately 12–14 kb (exact length to be confirmed by sequencing)

Vector Elements

Storage & Handling

• Plasmid Preparation: Use BioVector? endotoxin-free maxi-prep kit; amplify in BioVector? LB medium with ampicillin at 30°C to minimize recombination

• Storage: Short-term at -20°C; long-term at -80°C as glycerol stock or purified plasmid

Packaging & Transduction

• Packaging Cells: BioVector? HEK293T cells recommended

• Packaging Plasmids: Requires second- or third-generation helper plasmids (e.g., BioVector? psPAX2 and BioVector? pMD2.G)

• Transfection Reagent: BioVector? polyethylenimine (PEI) or BioVector? Lipofectamine? 3000

• Virus Harvest: Collect supernatant 48–72 hours post-transfection; filter through 0.45 μm membrane; may concentrate as needed

• Target Cells: Suitable for dividing and non-dividing cells, including BioVector? NHEK, BioVector? HEK293, BioVector? Jurkat, etc.

Features & Applications

• Efficient CRISPRi: Zim3 is a next-generation KRAB domain; fused with dCas9 it achieves potent and specific transcriptional repression with low off-target effects, ideal for loss-of-function studies

• Long-Term Stable Expression: The UCOE element resists position-effect variegation and promoter silencing, ensuring sustained CRISPRi activity in stem cells, primary cells, and in vivo models

• Reporter System: BFP fluorescent reporter allows real-time monitoring of transduction efficiency and expression; facilitates flow cytometry sorting and clonal selection

• Single-Vector System: All components integrated into one lentiviral vector, simplifying virus production and cell engineering workflows

• Key Applications:

  • Functional gene screening (CRISPRi libraries)

  • Inactivation of key genes in signaling pathways

  • Gene regulation in primary cells (e.g., T cells, neurons, keratinocytes)

  • In vivo delivery and gene therapy model development

Safety & Handling Notes

• This vector is a third-generation lentiviral system with SIN design; standard BSL-2 practices should be followed when handling virus

• Due to the presence of LTRs and repetitive elements, bacterial culture at 30°C is strongly recommended to avoid recombination

• Validate plasmid integrity by restriction digestion or sequencing before use