Making mice at high speed

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Laser-assisted injection of embryonic stem cells (ESCs) into 8-cell stage embryos yields mice that are fully ESC-derived.

Laser-assisted injection of embryonic stem cells (ESC) into 8-cell stage embryos yields mice that are fully ESC-derived.

In a recent cost-benefit analysis, the US National Institutes of Health (NIH) calculated that it takes a typical laboratory about one year to generate a transgenic mouse at a cost of one to two hundred thousand dollars.

These cost and time scales were prohibitive to a large-scale project the NIH announced in 2003: Knockout Mouse Project (KOMP), whose participants aimed to create ESCs with null mutations in every gene of the mouse genome and then generate a mutant mouse library from these ESCs. Two main technical hurdles stood between KOMP and its completion: the abilities to rapidly introduce deletions into ESCs on a genome-wide scale and to create a large number of transgenic mice from these ESCs in a reasonable time frame and for a reasonable cost.

Scientists at the biotechnology company Regeneron under the direction of David Valenzuela and George Yancopoulos decided to tackle both of these challenges. In 2003 they developed a technology to target genes in ESCs using homologous recombination to disrupt the endogenous gene with a bacterial artificial chromosome (Valenzuela et al., 2003). This allowed the researchers to rapidly modify ESCs but also left them with a dilemma. Yancopoulos describes it vividly, "We were deluged with ESCs that we could not make into mice with the original technologies." In a recent article in Nature Biotechnology, Valenzuela and Yancopoulos reported how they solved this problem with a new technique for rapidly generating transgenic mice from ESCs (Poueymirou et al., 2007).

Figure 1 | Comparison of two techniques to generate transgenic mice.

Full figure and legend

The scientists injected the ESCs into an early mouse embryo at the 8-cell stage, rather than into the blastocyst, a more mature embryo consisting of approximately 64 cells, which yields chimeric mice derived from both the ESC and host embryo. Their essential ingredient for success was a laser that they used to punch a hole into the zona pellucida, a membrane coat surrounding the embryo, before injecting the ESCs (Fig. 1). To their surprise and delight, they found that embryos injected with ESCs after laser puncture developed into mice that were wholly ESC-derived with virtually no contamination from the host embryo.

This combination of using 8-cell stage embryos and laser-assisted injection proved to have several other advantages over blastocyst injection. Eight-cell stage embryos are more uniform and robust than blastocysts, hence scientists need to kill fewer mice during the collection of the embryos. They found that 7–9 ESCs are sufficient for injection into the early embryo, fewer than required for a blastocyst, and ESCs from inbred strains, which often yield very poor results in blastocysts, can be used in early embryos. Perhaps most importantly, as the transgenic mouse is fully ESC-derived, it can be directly phenotyped, and no lengthy breeding is needed.

When asked about the technical challenges of this laser-assisted injection protocol, Valenzuela is confident that anybody who is proficient in microinjection will be able to use it. And he added that the cost of the laser will be amortized by the savings in time and reagents.

Although this injection technique will benefit individual laboratories, its main application is clearly in large-scale efforts such as KOMP, which need multilab collaborations and are impossible for a single lab to undertake. The good news for individual academic institutions is that they will not have to duplicate the effort; all mice created for KOMP will be freely available to them.

Nicole Rusk

References

Valenzuela, D.M. et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature Methods 4, 196–197 (2007). | Article | PubMed |