One Step Ahead with ingenious’ FLP ES Cell Lines
Selection cassettes such as Neomycin, Hygromycin, and Puromycin are commonly used to generate mouse knockout and knockin models. The cassette serves important purposes during the mouse model production process, but it may cause issues in later stages of mouse development and affect the phenotype , . It is recommended to remove the cassette before studying the model.
ingenious targeting laboratory has developed several proprietary FLP ES cell lines which aid to remove the selection cassette without any additional labor. This significantly speeds up the process of mouse model generation.
Here we discuss the utility of selection cassettes in mouse model development, define why they should be removed before studying the model, and provide examples of removal techniques.
Why is the Neo Selection Cassette Required?
In order to generate a mouse knockout or knockin model, recombinant embryonic stem (ES) cells with the correctly integrated targeting vector are injected into mouse embryos, which give rise to chimeric mice. To identify the correctly targeted ES cell clones, the selection cassette is required for two reasons:
After the targeting vector is introduced into an ES cell line, the cells are treated with a specific antibiotic based on the selection cassette that was used for the targeting vector. Thus, clones that survive the antibiotic treatment have the selection cassette integrated into the mouse genome.
Aid in Screening Strategies
The second reason for using the selection cassette is to provide a screening strategy to identify ES cell clones that have the correct integration of the targeting vector. The selection cassette provides unique sequences and restriction sites that are used to design PCR primers and Southern blot strategies, to identify and confirm clones that have the targeting vector integrated into the correct location in the mouse genome.
Because the the selection cassette may interfere with gene expression and negatively affect mouse development ,  it is recommended to remove the cassette before conducting studies with the mouse model.
How is the Neo Cassette Typically Removed?
The selection cassette is commonly flanked with FRT recombination sites, which are FLP recombinase recognition sites. In order to delete the selection cassette, the FLP recombinase enzyme must be introduced to recombine these FRT sites. This can be accomplished either in vitro via an additional electroporation step or in vivo via mating. In each case, additional labor is needed, resulting in longer project time lines and additional cost. Below we discuss both strategies in more detail, focusing on FRT/FLP recombination.
Additional Electroporation and Screening
The selection cassette can be removed right after the recombinant clones have been identified, with a second electroporation.
During the second electroporation, the site-specific recombinase FLP is introduced to delete the selection cassette. Chimera mice produced from those targeted, selection cassette-deleted ES cell clones are then mated with wild-type mice to produce the completed, germline confirmed mouse model.
There are drawbacks of using a second electroporation, however, such as the necessary extra manipulation and additional screening of the ES cells, longer project timeline, and additional labor required, and higher chances of reduced or absent germline transmission efficiency.
A traditional way of removing the Neomycin selection cassette in vivo is by mating chimeric mice to FLP transgenic mice to introduce the FLP recombinase. In this workflow, chimera mice produced from targeted ES cell clones still carrying the selection cassette are mated with FLP recombinase mice to initiate the selection cassette deletion process. At minimum, a subsequent round of mating with wild-type mice is required to complete the deletion process and produce the completed, germline confirmed mouse model.
The drawbacks of adding FLP mating to the model production workflow include having to purchase and maintain FLP mice, the extensive time, labor, and costs devoted to the additional breeding steps and genotyping, and the need for detailed tracking and record keeping of the mice.
ingenious’ FLP ES Cell Lines
Always striving to improve the mouse model production process, ingenious has created unique FLP ES cell lines that provide a labor-free introduction of the site-specific recombinase as soon as targeted ES cell clones have been identified.
How Does it Work?
Our FLP ES cell lines carry the FLP transgene which is ubiquitously expressed. After the targeting vector is introduced into one of our FLP ES cell lines, the recombinant clones carry both the selection cassette and the FLP recombinase. ES cell clone screening identifies targeted clones in which the FLP recombinase has deleted the selection cassette, and those clones are used to produce chimera mice. Those chimera mice are then mated with wild-type mice to produce the completed, germline confirmed mouse model. No second electroporation or additional mating steps are required for Neo removal.
The Advantages of Using Our FLP ES Cell Lines:
A shorter project timeline, with 3 to 6 months of saved time
Less manipulation of ES cells
Reduced animal husbandry needs and costs
Less genotyping work
Minimized human error
FLP ES Cell Lines Available:
Hybrid (C57BL/6 x 129/SvEv)
Learn more about our ES cell lines.
1) Hirotsune S, Fleck MW, Gambello MJ, Bix GJ, Chen A, Clark GD, Ledbetter DH, McBain CJ, Wynshaw-Boris A. 1998. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nat Genet 19:333–339.
2) Xu,X., Li,C., Garret-Beal,L., Larson,D., Wynshaw-Boris,A. and Deng,C.X. (2001) Direct removal in the mouse of a ¯oxed neo gene from a three-loxP conditional knockout allele by two novel approaches. Genesis, 30, 1±6