BacMam + multi gene expression approach

The protein interactome (or so-called multi-protein complexes) is reported to catalyze most of the key cellular functions (like replication, transcription, translation, transport processes across internal and external membranes, and signaling events, etc.) in mammalian cells. The multi-protein complexes typically consist of not only proteins but also nucleic acids and small molecules. To facilitate the protein interactome research, being able to express high-quality protein complexes is the prerequisite. A solution which has large capacity and support multiple gene simultaneous expression in mammalian cells is required.

BacMam is a hybrid system which facilitates heterologous DNAs delivery to mammalian cell using baculovirus. Baculovirus’s capacity to large size heterologous DNA makes it possible for mammalian cell to expression the protein interactome. As the conventional multi-expression process can be labor-intensive and inflexible, Creative BioMart provide a multi gene expression BacMam approach which support flexible insertion of multiple exogenous fragments into a wide range of mammalian cells.

Technology description

The basic principle MultiBacMam (see Figure 1) is similar to that of MultiBac. Find out more about MultiBac

• Several heterologous function cassettes were inserted into the vector (acceptor and donor), see Figure 2
• Vector recombines with viral DNA backbone by Tn7 transposon site in E. coli cells
• The recombinant bacmid was transfected into insect cells to synthesize the recombinant baculovirus.
• The recombinant baculovirus can be administrated to the selected mammalian cell line and guide the form and assembling of the desired protein interactome. 

Note. In order to be recognized by mammalian cells, strong viral/mammalian promoters (currently CMV and the hybrid CAG promoters) was added into the vector backbone previously.

Figure 1. The basic principle MultiBacMam

• The recombinant vectors can be constructed through either homing endonuclease (he) / BstXI multiplication or Cre-Lox recombination or standard fragment insertion via MCS. For the detail for these vector construction methods, please refer to our Generating Vectors Contains Multigene Expression Cassettes page.

Figure 2. How the multiple gene cassettes are inserted into acceptor (lift) and donor (right) vectors.

Features

Mini-Tn7 elements support the site-specific transposition between bacmid and vector

A CMV promoter from Cytomegalovirus is integrated into the BacMam vector to facilitate the gene expression in mammalian cells

Chloramphenicol resistance gene, Ampicillin resistance gene and Gentamicin resistance gene for vector selection

A vesicular stomatitis virus (VSV) peptide is integrate to the baculovirus genome previously to display the VSV peptide on the surface of the recombinant virus. Through this method, the mammalian cell transduction efficiency had been enhanced dramatically.

A mCherry fluorescent protein is also inserted into the backbone genome of baculovirus to convenient the monitoring of virus amplification with the pink color. Find out more about the baculovirus backbone

Service Content

Here are the service contents available at Creative BioMart, please click the link below for more information.

What Creative BioMart Provides for Our Customers

Baculovirus expression in mammalian cells can be achieved in a variety of pathways, each pathway has its own advantages and disadvantages, and cannot replace each other. Creative BioMart provides a comprehensive construction method of BacMam. Customers who are interested in our BacMam expression service, please click the links below to learn more detail about our services. For any question or request, welcome to contact us. 

• BacMam + Bac to Bac expression approach

An approach inherits construction principle and features of the Bac-to-Bac approach

• BacMam + Gateway expression approach

The BacMam vector is constructed via Gateway recombination