PSF-CMV-RAT-IGG1 HC - RAT IGG1 HEAVY CHAIN ANTIBODY PLASMID

This plasmid is designed for the generation and expression of heavy chain rat (Rattus rattus) immunoglobulin antibody genes. The vector has been formatted so that it can be cleaved to produce a restriction site overhang (using BseRI) within the first codon of the antibody constant region. This allows the variable regions of antibodies to be inserted to create full length heavy chain expression cassettes. As part of this product range we also have antibody vectors for the production of mouse and human antibodies as well as plasmids for the production of rat Kappa and Lambda antibody light chains (pSF-CMV-Rat-Kappa and pSF-CMV-Rat-Lambda respectively).

Promoter Expression Level: This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture.

The plasmid encodes a constant region an antibody in the main multiple cloning site positioned so that it can be cleaved to produce an overhang that allows seamless fusion with a variable region from any antibody. This allows you to create full length antibody genes with no cloning scars.

To enable this immediately upstream of the constant region coding sequence there is a BseRI restriction site. This is a type-IIS restriction enzyme that binds in one position (CAGCAG) and then cleaves a specific number of nucleotides away from the binding site regardless of the sequence at the cleavage point. We use this site in all of our antibody expression cassettes in the same position. In this plasmid cutting with BseRI will result in an overhang consisting of the first two nucleotides of the first codon of the constant region. This means that any variable region with the same overhang at its 3 prime end can be ligated into this plasmid when used in conjunction with any 5 prime site (NotI-NcoI). To add this overhang the variable region must be PCR amplified to contain any of the following sites at its 3 prime end: BseRI BsgI BtsI or BsrDI. By using this system it allows antibody variable regions to PCR amplified and fused to any of our constant region plasmids without having to re-synthesise the entire antibody expression cassette each time.

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