Imagine a poodle giving birth to a pomeranian. Like dog breeds, mouse strains can look and behave very differently. Researchers normally generate gene knockouts using embryonic stem cells and host embryos from two different mouse strains. The first generation (F0) of mice with the desired mutation carries a mix of genes from each mouse strain and therefore has a mottled coat, with patches of different colors. Now Poueymirou et al. report a gene knockout technique that generates F0 mice with genes only from embryonic stem cells, not the host embryo, in a recent article in Nature Biotechnology.
Researchers generate traditional knockout mice by injecting several male (XY) embryonic stem cells carrying a desired mutation into 200-cell embryos called blastocysts. Researchers breed F0 mice to females to generate heterozygous F1 mice and then breed F1 heterozygotes to each other to generate F2 homozygotes.
To increase the genetic contribution made by the embryonic stem cell to F0 mice, the authors injected embryonic stem cells into younger embryos. They perforated the outer membrane of eight-cell embryos with a laser and inserted a single XY embryonic stem cell. Mice in the resulting litter had either 100% host-derived or 100% embryonic stem cell-derived coat color. None of the mice had mottled coats. All of the mice with embryonic stem cell-derived coat color were F0 mice that expressed the desired gene mutation. The authors generated fertile female F0 mice using XO embryonic stem cells. Therefore, their technique generated mice homozygous for the desired mutation two generations earlier than the traditional technique.
The eight-cell embryo did not contribute genetically to F0 mice. The authors injected embryonic stem cells lacking the gene Il2rg into eight-cell embryos. In six different organs from F0 mice, the cycle threshold for Il2rg detection by TaqMan quantitative real-time PCR was greater than 40, indicating that the host embryo contributed less than 0.1% of the F0 genes, according to the authors. They injected blastocysts and eight-cell embryos expressing the lacZ reporter with embryonic stem cells lacking lacZ expression. They found lacZ-expressing cells in F0 mice derived from blastocysts, but not in F0 mice derived from eight-cell embryos.
Gene mutations generated by the eight-cell embryo technique can be phenotyped in F0 mice. The authors injected eight-cell embryos with XY and XO embryonic stem cells heterozygous for knockout of the insulin-like growth factor 1 gene. Both male and female F0 mice weighed less than wild-type mice, consistent with the phenotype of insulin-like growth factor 1 heterozygotes. Deletion of the delta-like ligand-4 is embryonically lethal. All F0 mice from eight-cell embryos injected with embryonic stem cells lacking delta-like ligand-4 died in utero. The authors believe that the ability to phenotype the F0 generation will facilitate high-throughput genomic studies.
Because blastocyst-derived F2 mice have mixed genetic backgrounds, many phenotypes found in F2 mice disappear when mice are backcrossed to genetic homogeneity, which generally requires 20 generations of breeding. In contrast, the eight-cell embryo technique produces genetically homogenous mice in the F0 generation, reducing both the time and money required to generate reliable knockout mice.