Multi gene expression approach is an innovation method based on the conventional Bac to Bac expression. One of the differences between two approaches is the gene cassettes insertion strategy. After all, multi gene insertion involves repeated restriction enzyme cleavage, thus not as easy as single gene insertion. The methods of exogenous gene insertion are flexible, rapid and simple in multi gene expression approach.
At Creative BioMart, three insertion strategies are available. Customs can make selection according to their own requirement or inquiry us for help.
There are always one or two multiple cloning site (MCS) on accepter and donor vector in Multi Gene Expression Approach. The MCS is flanked with a homing endonuclease (HE) site and a matching designed BstXI site (for acceptors vectors, I-CeuI is the homing endonuclease of choice, and for donor vectors PI-SceI). Standard method can be used to insert the first individual gene expression cassettes into the MCS. However, when there is already insert a cassette on vector, the HE site should be used to entire the following several cassettes. The homing endonucleases produce cohesive ends that are compatible with the ends generated by the BstXI digest.
The logic of HE multiplication is shown in Figure 2. Before inserting the Gene 2, the target vector is cut with the BstXI (at the position of green box). a homing endonuclease/BstXI hybrid restriction site is created that can then cannot be cut by either enzyme (crossed-out red/green box). Then the Gene 2 is connected to cleavage. The same process can be repeated over and over again to generate multigene assemblies.
Note. HE / BstXI multiplication is more suitable for multigene acceptor vector construction, although it also facilitates the multiplication of donor vector.
Cre recombinase is a member of the integrase family. Two DNA molecules which containing single LoxP sites can be fused under certain stimulation. The logic of Cre-Lox multiplication is shown in Figure 3. The gene cassettes are insert to vector via standard method or HE /BstXI cloning. The multigene as well as antibiotic resistance marker harbored in different vectors are then merged together into a single vector.
Both methods can also be combined to generate multiple gene-expression cassette constructs. The most commonly used procedure is to insert multiple gene cassettes with the HE /BstXI to acceptor and donor vectors and then fuse the vectors using Cre-Lox recombination.
We determine the price according to the complexity of the foreign gene insertion procedure.
| Number of Gene Cassette | Price | |
| 1 | 1-2 Gene Cassettes | Inquiry |
| 2 | 3-4 Gene Cassettes | Inquiry |
| 3 | 5-6 Gene Cassettes | Inquiry |
| 4 | 6-7 Gene Cassettes | Inquiry |
| 5 | 8 Gene Cassettes | Inquiry |
Want more gene cassettes insertion? Please contact us for quotations.