Re-establishment of a breeding colony of immunocompromised mice through revival of cryopreserved embryos.

Cryopreservation of embryos or germ cells is commonly used in animal facilities to archive strains of genetically modified mice, both to reduce facility space usage and to protect the strains from losses due to environmental disaster, genetic drift, disease outbreak or breeding failure. The authors' institution maintains a cryopreservation repository for various mouse strains, including immunocompromised mice. When the institution experienced a breeding failure with one strain of immunocompromised mice (NOD.CB17-Prkdc(scid)/NcrCrl), the authors successfully re-established a breeding colony of the mice by reviving frozen embryos from the institution's cryopreservation repository. They confirmed that the recovered progeny lacked T and B cells. The authors conclude that a breeding colony of immunocompromised mice can be successfully re-established from a minimal number of cryopreserved embryos.

Studying early lethality of 45,XO (Turner's syndrome) embryos using human embryonic stem cells.

Turner's syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos.

Developmental relationships between hemopoiesis and vasculogenesis.

Using avian chimeras, we have demonstrated earlier that the stem cells seeding the definitive hemopoietic organs form within the embryo rather than in the yolk sac. We now report experimental evidence indicating that hemopoietic progenitors appear in mouse embryos in the para-aortic splanchnopleura, a location similar to the one that produces stem cells in avian embryos. This structure obtained from E8.5 embryos was grafted under the kidney capsule of adult SCID mice. One compartment of the B lymphoid system became reconstituted with cells derived from the graft, that were identified with genetic and antigenic markers. In vitro data are also in favor of the production of progenitors from this structure. Finally a strategy designed to understand the developmental links between the endothelial network and hemopoietic cells is described. It is based on the expression patterns of two protooncogenes, c-ets1 and c-myb, activated during the amplification period of each of these lineages.