No one doubts that mice with a defective gene can reveal the origins of human diseases and show their progression, so much so that global consortia have been established to make lines of knockout mice for every mouse gene. However, the usual process of generating knockout mice lasts at least nine months and can continue for more than a year. A new laser-based method produces mutant mice that are ready for phenotypic analysis in about two months —a savings of at least six months. The standard means for making a mutant mouse is to introduce genetically altered mouse embryonic stem cells into mouse embryos at the blastocyst stage. This method produces chimeric mice that have some cells with the desired embryonic stem cell-derived genotype and some cells with the undesirable genotype of the embryo.To obtain mice with cells that all have the desired genotype, these chimeric mice must be bred for two generations — at a time cost of about three months per generation. In contrast, the laser-based method produces mice with the desired genotype in a single generation. David M. Valenzuela and colleagues at Regeneron Pharmaceuticals Inc. in Tarrytown, N.Y., developed the laser-based method.The researchers believed that if the embryonic stem cells were injected into the embryo at a stage that precedes the blasocyst stage — the eight-cell stage — the embryo might adopt more of the genotpe of the embryonic stem cells, providing a mouse that is genetically more desirable. However, the zona pellucida, a hard proteinaceous coat, surrounds the egg at the eight-cell stage, preventing the researchers from injecting stem cells into the embryo.To penetrate the zona pellucida, the scientists used the XYClone laser system from Hamilton Thorne Biosciences of Beverly, Mass. Valenzuela said that human fertility clinics commonly employ the same laser system to ablate a portion of the zona pellucida so that sperm can more easily penetrate the egg. Therefore, he and his colleagues decided to use the technology to improve mutant mouse production. Diarmaid Douglas-Hamilton, a principal developer of the laser system, said that it contains a proprietary InGaAsP laser diode incorporated into a 40× objective. The laser delivers a 1480-nm laser pulse and, because water surrounding the embryos strongly absorbs infrared radiation, the water is heated creating an opening in the zona pellucida without contacting it. Additionally, Douglas-Hamilton said that the short pulse is safer for the embryo than longer pulses.In their experiments, the scientists prepared the embryo for the injection of embryonic stem cells by making a perforation in the outer margin of the zona pellucida. They empirically determined the optimal laser settings. They used an 800-μs tangential pulse at 100 percent laser power (300 mW) to create an aperture large enough for the injection needle without damaging the embryo. The researchers used mouse embryonic stem cells from various genetic backgrounds, including the inbred C57BL/6 mouse strain. Valenzuela said that the consortia are using the embryonic stem cells from this mouse strain to make knockout mice.Uniform DNAThe laser-based method produced first-generation mice with a uniform coat color, in contrast to chimeric mice, which have a mottled appearance. The homogeneous coat color inspired the researchers to investigate whether the genetic transformation extended to the reproductive cells of the mice. Figure 1. The conventional method of genetically engineering mice involves injecting embryonic stem cells into blastocyst-stage embryos (a), but this produces mice with only part of the desired genotype. A laser system enabled researchers to insert the embryonic stem cells into mouse embryos at the earlier eight-cell stage (b,c), resulting in mice with the desired phenotype in a single generation. Images reprinted with permission of Nature Biotechnology.To do so, they crossed mutant mice with wild-type mice and determined the genotype of the offspring. They found that 96 percent of the mice transmitted their altered DNA to the next generation, whereas only 66 percent of the chimeric mice passed their mutant DNA on. To quantitatively determine the degree to which the embryonic stem cells contributed to the genotype of various tissues, the scientists performed real-time polymerase chain reaction (PCR) with a detector from Applied Biosystems of Foster City, Calif. They used real-time PCR to analyze lung, liver, heart, kidney, skeletal muscle and tail tissue. They found that the embryonic stem cells contributed more than 99.9 percent of their DNA to the knockout mice.As an additional quantitative measure, the investigators studied embryos expressing a lacZ reporter. They concluded that the mutant embryos contained less than 0.05 percent of the genetic material from the original embryo.Figure 2. Researchers injected embryonic stem cells into embryos expressing GFP. Because the embryonic stem cells (not expressing GFP) should colonize the embryo, the embryo should not fluoresce. As desired, embryos produced with the laser system did not fluoresce (right). However, the traditional method resulted in chimeric embryos with strong fluorescence signals (left).Next the investigators injected embryonic stem cells into embryos expressing enhanced GFP. Because the stem cells do not express GFP and they should colonize the embryo, the resulting mutant embryo should no longer fluoresce. The embryo generated by the laser-assisted method did not fluoresce at all, but the chimeric embryo strongly fluoresced (Figure 2). These quantitative analyses demonstrate that the laser-based method generates pure knockout mice within the first generation. The scientists detailed their experiment in the January issue of Nature Biotechnology.To understand why their technique works, the researchers studied a clump of cells inside blastocysts, called the inner cell mass, because embryonic stem cells are known to overtake those cells. They injected embryonic stem cells expressing GFP into embryos and compared blastocyst-stage embryos generated by the two methods. The inner cell mass uniformly expressed GFP in embryos produced with the laser-based method, but the conventional method resulted in blastocysts with a chimeric inner cell mass and scattered GFP expression (Figure 3).Figure 3. To understand why the laser-assisted method works, the scientists introduced embryonic stem cells expressing GFP into embryos using both the conventional and laser-assisted methods. The laser-based method allows the embryonic stem cells to colonize more of the inner cell mass region of the embryo, resulting in more fluorescence (left) than produced by the traditional method (right).Valenzuela said that when the conventional method is used, only male mice are useful in the first generation because of the nature of the technique. However, he said that the laser-based method also can produce first-generation female mice, which can be bred with their male counterparts to save three months. With the laser-assisted method, the scientists created female knockout mice in the first generation by using male embryonic stem cells previously selected for the loss of their Y chromosome. The resulting female mice were healthy and fertile.The scientists used the laser-assisted technique to create three well-characterized lines of knockout mice and then examined them. The first line did not have the gene that encodes insulinlike growth factor-1, and the absence of this gene led to a decrease in body weight. The second line was missing the gene that encodes IL2Rg, and lacking this immune system gene causes severe combined immunodeficiency, commonly called bubble-boy syndrome in affected humans. The third line did not contain Dll4, a developmental gene for delta-like ligand-4, without which vascular defects appear, causing all knockout mice to die at birth. In all cases, the scientists observed the expected phenotype. These three mice lines show that the laser-based method can produce mutant mice that are ready for phenotypic analysis in a single generation.As a result of the saved breeding time, Valenzuela said, “The cost savings would be enormous.” He estimates that using the laser-based technique to inject eight-cell-stage mouse embryos will double the efficiency of personnel and cut operating costs in half. Regeneron markets two of the techniques used to generate the knockout mice in this research. Its VelociGene and VelociMouse methods rapidly produce genetically engineered embryonic stem cells and mice, respectively.Contact: David M. Valenzuela, Regeneron Pharmaceuticals Inc., Tarrytown, N.Y.; e-mail: info@velocigene.com; Diarmaid Douglas-Hamilton, Hamilton Thorne Biosciences Inc., Beverly, Mass.; e-mail: kbradley@hamiltonthorne.com