More efficient mouse model generation through informed founder selection

Lab mouse

This paid-for advertorial by Taconic appeared in DDW Volume 24 – Issue 3, Summer 2023

Overcoming challenges in model generation

Genetically engineered mouse models are essential tools for biomedical research. Yet when seeking to obtain an existing model, researchers often encounter obstacles related to intellectual property, availability, or licensing. As a result, model generation is an attractive option often considered in parallel. However, the creation of custom mouse models remains a complex process that comes with significant risk(s).

This editorial focuses on the importance of informed founder selection and highlights a data-driven approach that significantly de-risks the process of germline transmission.

The significance of germline transmission

Germline transmission is the successful inheritance of an embryonic stem cell (ESC)-derived allele, present in a founder, by its offspring. This is a critical step in model generation, as founders are not only genetically complex but vary individually in several important ways.

For projects that utilise gene targeting in ESCs, founders are chimeric (composed of genetically distinct cells derived from two different sources: modified ESCs and a host blastocyst). Host embryos from a different inbred strain are traditionally used. This results in distinct coat colour patterns in each chimera, indicating the relative contributions of the ESC and host blastocyst components.

Coat colour patterns are traditionally used to make an inference about the presence of the modified allele in the germline. However, it is impossible to verify this without direct assessment, as the levels of chimerism may vary across different tissues. Hence, coat colour scoring alone leaves room for both false positive and false negative judgements about the potential for germline transmission.

In response, researchers often breed several founders. Additional issues can also be discovered at this stage, however, including sterile and/or hermaphroditic founders – highlighting the unrelated risk of relying on breeding or natural expansion for germline transmission.

Informed founder selection for de-risking germline transmission

A comprehensive approach to founder selection can mitigate these risks, and extensive molecular characterization of founders plays a crucial role in that process.

By harvesting sperm from candidate male founders and utilising qPCR, it is possible to detect the modified allele and estimate its percentage in the germline. An example project (Fig. 1A) demonstrates the potential for poor correlation between coat colour and direct assessments of germline transmission potential. In Chimera 2, the percentage of the humanized allele in the sperm (≥ 50%) is consistent with a near-total contribution from the ESC component (a heterozygous ESC clone). In turn, 100% of the offspring were derived from the ESC component, and approximately 50% of those contained the modified alleleas expected (Fig. 1B).

If a chimera was selected solely on the basis of coat colour, a dramatically different result could have been observed. The requisite sperm cryopreservation and in vitro fertilisation (IVF) procedures involved also help avoid any potential issues related to sterility or reproductive performance.

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Figure 1: Characterisation of chimeric founders for a genetically humanized allele. (A) Comparison of founder selection methods: coat colour vs. qPCR measurement. (B) 100% germline transmission from a chimeric founder selected using a direct assessment of candidate founder sperm.

Applying informed founder selection to CRISPR model generation

Similar techniques can also be applied to embryo-based model generation technologies such as CRISPR/Cas9 gene editing, to assess both on-and off-target editing in founders. An in-depth discussion of this method is, however, outside the scope of this editorial.

Reducing time, cost, and effort

A data-driven approach enables the identification of founders with the highest germline transmission potential and helps to secure project timelines. It also helps facilitate scaling of germline transmission efforts, further reducing the time to meaningful cohorts.

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