The generation of genetically modified mice may be divided into two main categories: targeted integration (insertion or deletion) and random integration of DNA into the mouse genome. Gene targeting is typically performed in 1 of 2 ways: 1. embryonic stems (ES) cells are ideally suited to larger DNA insertions (or modification) of endogenous alleles. 2. The MBP also uses newer genome editing technologies (e.g., CRISPR/Cas) to create knockout and knockin mice at lower cost and in significantly less time than conventional, homologous recombination-based approaches in ES cells. Random integration is generally performed by pronuclear injection or electroporation to generate transgenic mice by random integration of DNA (e.g., LacZ, GFP, etc) into the mouse genome.
Your specific scientific hypothesis-driven question will drive which approach to use to create the most appropriate mutant mouse model for research.
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|ES cell-targeted Models|
|Site specific integration, no unintended disruption of endogenous gene function.||Increased time to generate founders due to cell culture and germline transmission from chimeras.|
|Single copy insertion of gene construct, rarely undergoes gene silencing.||Higher overall project cost of compared to random transgenics and CRISPR.|
|Stable integration and germline transmission.||Limited to genetic background of available, germline competent, ES cells.|
Multiple independent founders may be necessary to identify the desired expression pattern.
|Lower overall project cost compared to ES cell based targeting.||Unpredictable insertions sites. Endogenous gene disruption that may lead to confounding phenotypes.|
|Rapid generation of founders.||Multiple insertion sites and/or copy number leading to diminishing expression or silencing.|
|Not limited by genetic background|
|Lower overall project cost compared to ES cell based targeting.||Low flexibility in model design and transgene insertion size.|
|Shorter project timeframe. In general, KO creation using CRISPR/Cas9 can create an InDel in 1/3 the time of traditional ES cell targeting||Off target effects need to be identified and bred out in subsequent generations|
|Not limited by genetic background. The MBP has high success using the CRISPR/Cas9 platform in C57BL/6 genetic backgrounds|