Gateway Entry Clones

• RNAi expression—Efficient delivery and long-term stable or transient shRNA expression in any mammalian cell types with Lentiviral and Adenoviral Gateway vectors
• Viral expression—High level, stable and transient gene expression in any mammalian cell type
• In Vitro protein synthesis—Directly synthesize high yields of recombinant protein in a single reaction tube in just 2 hours without special equipment. Scale the reaction to achieve microgram to milligram levels of protein
• in vivo biotinylation—Easy efficient method for expressing, purifying and detecting biotinylated recombinant proteins.
• Tag-On-Demand technology—Produce native or tagged proteins from one vector.
   

The three possible methods that lead to the Entry clone are depicted in Figure 1. Using TOPO vectors and PCR amplification/restriction-enzyme vectors are the most common ways to construct your own Entry clone.

Figure 1. Strategies to build the Entry clone. The three possible methods that lead to the Entry clone are depicted: (A) BP cloning, (B) TOPO cloning, and (C) restriction enzyme and ligase cloning. Red arrows represent the fragment of interest. Adapted from Katzen F (2007) Expert Opin Drug Discov 2(4):571–589.

We offer two types of TOPO vectors that offer easy TOPO cloning to create a Gateway Entry vector. pCR8/GW/TOPO vector kits and pENTR/D-TOPO vector families both offer 5-minute, efficient TOPO cloning, with >95% efficiency.

pENTR/D-TOPO vectors take advantage of fast, efficient Directional TOPO cloning that delivers your insert in the correct orientation for expression. These vectors contain the necessary attL sequences for recombination into any Destination vector and certain versions carry a TEV protease cleavage site for producing native proteins after expression (Figure 2).

Figure 2.Several pENTR vectors are available for Directional TOPO cloning and direct access to the multitude of Gateway expression vectors.

The pCR8/GW/TOPO vector enables efficient TOPO-TA cloning. These vectors easily facilitate multipurpose use: rapid recombination into a variety of Gateway Destination vectors, convenient sequencing, robust selection in E. coli with spectinomycin resistance, and easy excision of insert DNA with flanking EcoR I sites (Figure 3).

Figure 3.The pCR8/GW/TOPO Entry vector allows TOPO TA Cloning for multiple downstream applications.