pcDNA3.3-TOPO载体： The pcDNA3.3-TOPO vector is a TOPO-adapted plasmid that allows rapid cloning of a PCR product containing a gene of interest downstream of the CMV promoter. The pcDNA3.3-TOPO vector can be used for high level expression of a native protein in adherent mammalian tissue culture cells following transient transfection, or high level expression of secreted, native protein using the FreeStyle MAX CHO and FreeStyle MAX 293 systems.
The pcDNA3.3-TOPO vector is also included with the OptiCHO Antibody Express System for cloning the light and heavy chains of your antibody of choice. 载体特征： Full-length human cytomegalovirus (CMV) immediate-early promoter/enhancer for high-level gene expression in a wide range of mammalian cells
TOPO Cloning site for rapid and efficient cloning of Taq-amplified PCR products
Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript
Neomycin resistance gene for selection of stable cell lines with Geneticin
pUC origin for high copy replication and maintenance of the plasmid in E. coli
Ampicillin (bla) resistance gene for selection in E. coli
CMV Promoter
The human cytomegalovirus immediate-early (HCMV IE1) gene promoter in the pcDNA3.3-TOPO vector is 680 bp and contains the native transcriptional start site (Hennighausen & Fleckenstein, 1986). This sequence results in high levels of transgene expression. TOPO技术简介： The pcDNA3.3-TOPO vector is supplied linearized with:
Single 3′ thymidine (T) overhangs for TA Cloning
Topoisomerase covalently bound to the vector (this is referred to as“activated” vector) Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3′ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO Cloning exploits this reaction to efficiently clone PCR products.Once the PCR product is cloned into the pcDNA3.3-TOPO vector and the transformants are analyzed for correct orientation and reading frame, the expression plasmid may be transfected into your cell line of choice. 实验流程： To TOPO Clone your gene of interest into pcDNA3.3-TOPO, you will perform the following steps:
1. Generate a PCR product containing your gene of interest with Taq polymerase.
2. TOPO Clone your PCR product into the pcDNA3.3-TOPO vector and use the reaction to transform One Shot TOP10 Chemically Competent E. coli.
3. Pick colonies, isolate plasmid DNA, and screen for insert directionality by sequencing expression clones with the primers provided in the kit. 建立稳定细胞系： After creation of your four clones, you will then use the OptiCHO Antibody Express Kit to transiently transfect two of each of the plasmid constructs (one of each pcDNA3.3 and pOptiVEC) containing the heavy and light chains into CHO cells. You may also perform several stable transfections directly in DG44 cells with various combinations of pOptiVEC-TOPO and pcDNA3.3-TOPO plasmid constructs.
