Design and Construction of Custom Targeting Vectors
Disruption of a gene in the mouse genome can be achieved by replacing it with its mutant/modified version created in a plasmid vector called the targeting construct. At the mouse genome engineering core facility, we design and generate gene targeting constructs. We use many cloning methods including “recombineering” to generate wide variety of targeting constructs for Cre-lox based conditional knockout or targeted knock-in vectors. We also generate conventional knockout and transgenic constructs. The details of constructing the conditional knockout vector, a most common type of vector, are described below*. The fee for custom (gene specific) conditional targeting vector construction is $5000**.
The project involves multiple steps and these steps are categorized under various stages and sub stages as outlined below
- Designing of the strategy
- Designing of the targeting vector [analysis to make decision about exons to delete, length of 5’ and 3’ arms, location of LoxP recombination sites etc.]
- Designing of the Southern blotting and/or PCR screening assay
Establishing of the Southern screening method is done at the PI’s lab; MGECF will help in troubleshooting if any)
We will discuss the targeting strategy with the Investigator for their approval before we begin work. Our expected timeframe for vector construction is approximately 3 months or less and all efforts will be made to complete well before 3 months! The fees will be billed stage wise after completion of each stage or at the end of last stage.
- Identification and Procuring of the BAC clone (or amplifying/synthesizing the fragments from genomic DNA, if feasible)
- Expansion and testing of the BAC clone
- Construction of the targeting vector
- cloning the outermost boundaries of the genomic region to be swapped into a plasmid backbone
- Recombineering [Gap repairing-actual swapping of genomic region from BAC into plasmid]
- Construction of minitargeting vector to insert first LoxP site
- Recombineering [First LoxP insertion]
- Construction of minitargeting vector to insert first second LoxP and Neomycin sites
- Recombineering [Frt-Neo-Frt-second LoxP insertion]; the final construct
- Testing of the targeting vector
- Sequencing vector insert junction
- Verification of functionality of Frt site
- Verification of functionality of LoxP site
The process of designing and construction of targeting vectors is highly complex and it involves careful consideration of several factors. To ensure that all possible factors (gene location, its promoter, adjacent genes intron/exon spacing, domains etc.) are taken into consideration, such projects require multiple sessions of meeting of the MGECF staff with PI (or PI’s lab personnel) to offer teaching and consultation sessions- which is extremely critical. Furthermore, each targeting construct (conditional KO or KI) is unique: in order to best serve the needs of the investigator, it necessitates us in understanding the biological significance of the desired mutation, going through the preliminary data and also reading of the published literature about the gene. A significant amount of time is spent in such activities and therefore we request that the targeting vector design and construction projects should be considered as collaboration.
* Combined knockout and knock-in strategies can also be designed in certain cases. Additional cost involved in designing and building such custom cassettes will depend on the construct design and project needs.
** Targeted knock-in into well-established loci such as ROSA 26 can also be designed at cheaper costs that depend on the complexity and/or availability of the cassettes to be inserted and the number of cloning steps involved.
Centers & Core Facilities
Bioinformatics and Systems Biology Core
- DNA Sequencing
- Electron Microscopy
Experimental Irradiation Facility
Mouse Genome Engineering Core
- Starting a Project
- Bioinformatics and Systems Biology Core
Advanced Anatomy Laboratory
- Research Labs