pCDH Vectors

This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles.

This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on SBI’s website. Before using the reagents and material supplied with this system, please read the entire manual.

B. Advantages of the Lentivector Expression System

Lentiviral expression vectors are the most effective vehicles for the delivery and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols.

However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides the stable, long-term expression of the target gene. SBI offers a third generation of the most popular HIV-1 based lentivector expression system which consists of three main components:

(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)

(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)

(3) A pseudo viral particle producer cell line (e.g., 293TN cells)

The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles.

To produce a high titer of viral particles, expression and packaging vectors are transiently co-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system, please refer to the Lentivector Expression System user manual.

C. pCDH Cloning and Expression Lentivectors

SBI provides a collection of cDNA cloning and expression vectors for various applications (Tables 1-4). For optimal results, we highly recommend packaging the lentiviral vectors into pseudo viral particles for transduction into target cells. Transfection of the plasmid into cells, while functional, may not be indicative of actual gene expression as there is an upstream constitutive promoter (RSV) that drives expression of all transgenes downstream.

For all pCDH lentivectors, there is a limit for the size of the insert(s) that can be cloned into the vectors for efficient packaging into pseudo viral particles. Typically, a pCDH lentivector can hold up to 3-5kb of insert (depending on the vector) such that the total 5’ LTR to 3’ LTR size including the insert is ~9.5kb.

1. Single Promoter Vectors

These lentivectors are characterized by the presence of a single mammalian promoter driving the gene on interest cloned into the MCS, either expressed by itself or co-expressed with a marker gene of interest (e.g. copGFP or Puro) in a T2A format (please see Section I.D). Vector maps and additional information can be found here:

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