Della Yee

A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes.

Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.

Genetic deletion of a neural cell adhesion molecule variant (N-CAM-180) produces distinct defects in the central nervous system

N-CAM is abundantly expressed in the nervous system in the form of numerous structural variants with characteristic distribution patterns and functional properties. N-CAM-180, the variant having the largest cytoplasmic domain, is expressed by all neurons. The N-CAM-180-specific exon 18 has been deleted to generate homozygous mice unable to express this N-CAM form. The most conspicuous mutant phenotype was in the olfactory bulb, where granule cells were both reduced in number and disorganized. In addition, precursors of these cells were found to be accumulated at their origin in the subependymal zone at the lateral ventricle. Analysis of the mutant in this region suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway. Subtle but distinct abnormalities also were observed in other regions of the brain.

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles. Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes,. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics-from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

Short-term rescue by RNA injection of a mitotic arrest mutation that affects the preimplantation mouse embryo.

The mutation oligosyndactyly results in syndactyly, abnormal fusion and insertion of certain limb muscles, and diabetes insipidus in heterozygous mice. When homozygous the mutation is lethal; beginning at the blastocyst stage, the homozygous cells arrest in metaphase with intact spindles. The mutant phenotype cannot be corrected by forming aggregation chimeras with wild-type cells, suggesting that the mutation results in a cell autonomous lethal condition. Short-term rescue of the homozygous-induced mitotic arrest can be achieved, however, by cytoplasmic injection of polyadenylated RNA obtained from a rapidly dividing embryo-derived stem cell line.

Epidermal Growth Factor Receptor Deficiency Results in Periimplantation Lethality in Mouse

The gene Egfr that encodes the epidermal growth factor receptor (EGF-R) is activated at the 8-cell stage of preimplantation mouse development (1). At the blastocyst stage the receptor is tightly regulated by the maternal steroid hormone status and has been found to be expressed on trophoblast cells, and to a lesser degree, on inner cell mass (ICM) cells (2). The receptor is also present in uterine tissues before and after implantation, with levels increasing during decidualization (3). After implantation, EGF-R is expressed in a wide variety of tissues of the developing fetus (4).