Absent forebrain replaced by embryonic stem cells

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For the past few decades, it has been possible to directly manipulate an organism’s genes early in development, to generate, for example, mice that harbour genetic modifications. Such transgenic mice have been powerful model systems in which to study human disease1, but conventional approaches for generating these animals can be costly and time-consuming. Writing in Nature, Chang et al.2 describe an alternative approach to building complex mouse models with which to interrogate the function and diseases of the forebrain. The authors’ approach could also be used to produce models focused on other regions of the central nervous system (CNS).

In conventional transgenic approaches, genes are modified in mouse embryonic stem (ES) cells, which are pluripotent — they can give rise to all cell types of the animal’s body. The genetically engineered cells are then injected into a mouse embryo at an early stage of development called the blasto-cyst stage. The result is a chimaeric mouse, in which some cells are genetically modified and some are not. If the animal’s eggs or sperm contain the genetic modification, it can be bred to produce offspring in which all the cells are modified (Fig. 1a). This approach, although recently accelerated by gene-editing technologies such as CRISPR–Cas9, remains expensive and labour-intensive. Moreover, unless the genetic modification is engineered to be expressed in only certain cell types, this approach is limited to genes that are not essential for early embryonic development.

Diagram of two ways to generate genetically modified mice.

Figure 1 | Two ways to generate genetically modified mice. a, Genetically modified mice are usually generated by engineering mouse embryonic stem (ES) cells to harbour a desired mutation, and injecting the modified cells into a host embryo at the blastocyst stage. The embryo is then implanted into a foster mother. The donor and host ES cells mingle, giving rise to chimaeric mice consisting of both cell lineages (indicated by colouring). By breeding with wild-type mice, chimaeric animals can pass the mutation down through their eggs or sperm, producing offspring in which all cells are genetically modified. b, Chang et al.2 describe an alternative approach called neural blastocyst complementation to produce mice whose forebrain cells all carry a desired genetic modification. The authors generated host embryos in which diphtheria toxin subunit A was expressed only in forebrain progenitor cells — these cells therefore died, leaving a vacant niche. The group injected the host blastocysts with donor ES cells that harboured a genetic modification of interest, and implanted them into foster mothers. The donor cells filled the vacant niche, repopulating the forebrain (but not other brain regions) of the resulting mice.

More than two decades ago, the laboratory that performed the current study devised a technique called blastocyst complementation, to circumvent these limitations in the immune system3. The approach is based on the fact that, if the development of a particular organ in the host is disabled, a vacant niche is created that can be filled by tissue derived from newly introduced ES cells. In that paper, the authors used RAG2-deficient mice, which do not have mature immune cells called T and B cells. They showed that embryos of this strain could give rise to mice that generated T and B cells normally if blastocysts were injected with wild-type mouse ES cells. Moreover, these immune cells were exclusively of donor origin.

Blastocyst complementation has since been used to generate the lens of the eye4, the kidney5 and the heart6. The technique has also been used to generate pancreases made mainly of cells from another species, by injecting mouse cells into pancreas-disabled rat blastocysts and vice versa7. This type of animal, called an interspecies chimaera, offers great potential both for understanding fundamental principles in biology and for regenerative medicine, in which the ultimate goal is to grow organs that carry the genome of a specific individual.

Chang et al. developed an approach that they call neural blastocyst complementation (NBC), which involved disrupting the developing mouse forebrain — the region that will generate the brain’s hippo-campus, cerebral cortex and olfactory bulbs, among other structures (Fig. 1b). They crossed existing transgenic mouse strains to produce embryos in which diphtheria toxin subunit A was expressed specifically in forebrain progenitor cells. This led to the death of these cells, and therefore to mice that lacked forebrain structures. But wild-type ES cells injected into blastocysts of this strain could repopulate the forebrain niche. The authors found that the forebrain structures in the resulting animals were reconstituted. These mice were indistinguishable from controls in a set of behavioural assays.

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