The pressure to produce results quickly during the drug discovery and development process continues to increase as does the role of genetically engineered custom mouse models. However, even the fastest custom mouse model generation projects take about 6 months to reach the stage when a few F1 heterozygous mice are available for experiments or breeding. This timeline increases if more than a few heterozygotes or homozygotes are needed. Taconic’s ExpressMODEL® portfolio of products shifts the deliverable of a model generation project from a few heterozygous F1 mice to a much larger cohort of 10-100 mice while reducing the timeline to obtaining data, adding predictability, all without compromising essential quality control steps. By applying innovative thinking and leveraging our ability to seamlessly integrate custom model generation, embryology and colony management services, the ExpressMODEL® portfolio achieves the industry's fastest timelines to study cohort with no compromise in quality for models generated using embryonic stem cell (ESC), CRISPR, or random integration transgenic (RITg) methodology.
Regardless of the methodology used to generate the founder animals, ExpressMODEL® is built around the concept that using in vitro fertilization (IVF) rather than conventional breeding to generate the F1 mice from founders has a number of distinct advantages including:
- Predictable timelines that are not dependent on natural breeding.
- Synchronized production of the F1 cohort.
- Customizable F1 cohort size to meet the customer’s needs.
- No need for an extra round of expansion breeding, thus saving at least 12 weeks from the time when a study-size cohort of heterozygous or homozygous mice is available.
- Possibility to generate the colony at any health standard.
However, ExpressMODEL® is more than simply using IVF to produce F1 mice because the quality of the male founders needs to be high in order to fully realize the advantages IVF provides. High quality means that founder males need to have both a high percentage of the desired genetically-modified gene and a high fertility rate. Thus, the candidate founder males need to be well-characterized. Because the different methodologies used for model generation produce founders with different characteristics, we have developed unique founder analysis protocols to fit the three different methodologies. The founders produced from injection of ESCs into blastocyst-stage embryos are called chimeras because they are derived from two different populations of genetically distinct cells that originated from different embryos. The founders produced by introducing CRISPR reagents or transgenic DNA into one-cell embryos (zygotes) are mosaics meaning they are composed of two or more different populations of genetically distinct cells that originated from the same embryo.
ExpressMODEL®: Embryonic stem cell (ESC)
ESC-mediated mouse model generation remains the gold standard and best choice for complex projects such as genomic replacement humanizations. Using ExpressMODEL®: ESC, the timeline for a typical project that would take 66 weeks to deliver a homozygous study-size cohort is reduced to 54 weeks, saving at least 3 months. The key components of ExpressMODEL®: ESC are:
- Additional fully validated ESC clones are produced and microinjected into embryos to ensure that clones with good germline potential are identified.
- Sperm is harvested and cryopreserved from male chimeras, and fertility rate is determined.
- Sperm DNA is analyzed by qPCR to determine the percentage of sperm cells carrying the modified allele directly, rather than relying only on the traditional indirect method of assessing ESC contribution to the germline by visually assessing the contribution of ESCs to the fur by inspecting coat color.
The data from these analyses facilitate the choice of founder male(s) to be utilized for the IVF to produce an F1 heterozygous cohort that is sized to meet the customers downstream goals and timeline requirements. It is important to note that all quality control steps in vector construction and ES cell targeting are preserved.
ExpressMODEL®: CRISPR
Two great advantages of CRISPR methodology are the speed at which a genetically engineered model can be generated and the ability to modify a wide range of genetic backgrounds, including existing genetically-engineered models. Using ExpressMODEL®: CRISPR, the timeline for a typical project that would take 48 weeks to deliver a homozygous study-size cohort is reduced to 36 weeks, saving at least 3 months. ExpressMODEL®: CRISPR combines our ability to produce founders with a low degree of mosaicism and to accurately estimate the degree of mosaicism of each founder male. The key components of ExpressMODEL®: CRISPR are:
- The on-target site and potential predicted off-target sites are assessed in male founders using next-generation sequencing of amplicons to determine the degree of mosaicism.
- This mosaicism data is used to select the best founder to use to establish a line using IVF.
- The F1 heterozygous offspring are analyzed using next-generation sequencing of amplicons to ensure that the delivered mice share the same molecular allele at the on-target site and to determine if any have inherited a mutation at a potential predicted off-target site.
ExpressMODEL®: Random Integration Transgenic (RITg)
More than 30 years since the first RITg model was generated, the method continues to be a favored path to quickly generate “gain of function” models that express an ectopic gene. However, because genomic integration of the transgene is random in each injected embryo, the resulting founder line are unique and may or may not perform to the desired specifications. Additionally, each founder often has transgene insertions at multiple sites and the configuration and copy number of those insertions differs. Thus, RITg founders can be more genetically complex than CRISPR founders. As a result, common practice is to separately propagate multiple lines to generate offspring for extensive transgene expression studies. These data are then used to determine which founder line(s) to propagate. The cost and time of this downstream breeding and characterization of multiple founder lines greatly exceeds the original cost to generate the lines and takes significant additional time. Because transgene expression is assessed in founder animals, ExpressMODEL®: RITg takes the guesswork out of the process and allows one to avoid the cost of breeding and characterizing multiple founder lines. Moreover, it reduces project timeline by at least 12 weeks and potentially up to 24 weeks or more. The key components of ExpressMODEL®: RITg are:
- Sperm is harvested and cryopreserved from male founders, and fertility rate is determined.
- Transgene copy number is determined by qPCR analysis of sperm DNA
- Transgene expression in specified tissue is determined by qRT-PCR analysis
- Fertility rate, copy number and expression data is used to determine the best founder(s) to use to establish a line
Additional customizable options are available including the provision of tissue lysates and fixed tissue for protein expression analyses, and transgene mapping analysis to accurately determine the transgene integration site and configuration.
Taconic’s ExpressMODEL® suite of technologies is designed to reduce the custom model generation timeline from project conception to study cohort without taking any shortcuts that compromise quality. Taconic provides a seamless end-to-end solution incorporating industry leading model generation, embryology, and colony management capabilities that allows your project to travel in the express lane.