Martin L. Hooper

Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells

The deletion mutation in the HPRT-deficient mouse embryonic stem (ES) cell line E14TG2a has been corrected by gene targeting. The presence of plasmid sequences in the correcting vector DNA did not affect the frequency of correction. We have characterized three different HPRT gene structures in correctants. Cells from one corrected clone have been introduced into mouse blastocysts, and germ line transmission of the ES cell-derived corrected gene has been achieved. The corrected gene has the same pattern of expression as the wild-type gene, with the characteristic elevated level of expression in brain tissue. Hence, we have demonstrated the feasibility of introducing targeted modifications into the mouse germ line by homologous recombination in ES cells.

Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells

Many pluripotent embryonal carcinoma (EC) cell lines and all embryonic stem (ES) cell lines have hitherto been maintained in the undifferentiated state only by culture on feeder layers of mitomycin C-treated embryonic fibroblasts. We now demonstrate that medium conditioned by incubation with Buffalo rat liver (BRL) cells prevents the spontaneous differentiation of such cells which occurs when they are plated in the absence of feeders. This effect is not mediated via cell selection but represents a fully reversible inhibitory action ascribed to a differentiation-inhibiting activity (DIA). BRL-conditioned medium can therefore replace feeders in the propagation of homogeneous stem cell populations. Such medium also restricts differentiation in embryoid bodies formed via aggregation of EC cells and partially inhibits retinoic acid-induced differentiation. The PSA4 EC line gives rise only to extraembryonic endoderm-like cells when aggregated or exposed to retinoic acid in BRL-conditioned medium. This suggests that DIA may be lineage-specific. DIA is a dialysable, acid-stable entity of apparent molecular weight 20,000-35,000. Its actions are reproduced neither by insulin-like growth factor-II nor by transforming growth factor-beta. DIA thus appears to be a novel factor exerting a negative control over embryonic stem cell differentiation.

Somatic frameshift mutations in the MBD4 gene of sporadic colon cancers with mismatch repair deficiency

Defects of mismatch repair are thought to be responsible for carcinogenesis in hereditary non-polyposis colorectal cancer and about 15% of sporadic colon cancers. The phenotype is seen as microsatellite instability and is known to be caused either by mutations in mismatch repair genes or by aberrant methylation of these genes stabilizing their downregulation. Lack of repair of microsatellite sequence errors, created during replication, leads to a mutation-prone phenotype. Where mutations occur within mononucleotide tracts within exons they cause translation frameshifts, premature cessation of translation and abnormal protein expression. Such mutations have been observed in the TGFβRII, BAX, IGFIIR, MSH3 and MSH6 genes in colon and other cancers. We describe here frameshift mutations affecting the gene for the methyl-CpG binding thymine glycosylase, MBD4, in over 40% of microsatellite unstable sporadic colon cancers. The mutations all appear heterozygous but their location would ensure truncation of the protein between the methyl-CpG binding and glycosylase domains, thus potentially generating a dominant negative effect. It is thus possible that such mutations enhance mutation frequency at other sites in these tumours. A suggestion has been made that MBD4 (MED1) mutations may lead to an increased rate of microsatellite instability but this mechanism appears unlikely due to the nature of mutations we have found.

While K-ras Is Essential for Mouse Development, Expression of the K-ras 4A Splice Variant Is Dispensable

ABSTRACT In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras tmΔ4A/+ mice produced viable K-ras tmΔ4A/tmΔ4A offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras tmΔ4A/tmΔ4A mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola × C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.

Towards the isolation of embryonal stem cell lines from the sheep

SummaryImmunosurgical isolation of inner cell masses (ICMs) from sheep embryos was most efficient at the expanded, zona-intact blastocyst stage (day 7 to 8 post oestrus) before migration of endoderm cells beyond the boundary of the ICM across the blastocoelic surface of the trophectoderm. When cultured under conditions which allow the isolation of embryonal stem (ES) cell lines from mouse ICMs, sheep ICMs attached, spread and developed areas of both ES cell-like and endoderm-like cells. After prolonged culture only endoderm-like cells were evident. The implications for the isolation of ES cell lines from sheep embryos and possible species-specific requirements are discussed.

Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line

SummaryThe derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.

Medium conditioned by feeder cells inhibits the differentiation of embryonal carcinoma cultures

Non-dividing STO mouse fibroblasts have been used for some time as feeder cells for maintaining certain embryonal carcinoma cells in an undifferentiated state. We report here that medium conditioned by these feeders can inhibit embryonal carcinoma (ec) cell differentiation induced either by removal from feeders, or, in the case of cells not normally requiring a feeder layer, by retinoic acid treatment.

The role of the p53 and Rb-1 genes in cancer, development and apoptosis.

SUMMARY Gene targeting using embryonal stem cells has been used to generate strains of mice with inactivating mutations at the Rb-1 and p53 tumour suppressor loci. Mice heterozygous for a null allele of Rb-1 do not show retinoblastomas but instead develop pituitary tumours. Homozygotes die at between 10 and 14 days’ gestation and show increased levels of both cell division and cell death by apoptosis in the haematopoietic and nervous systems. This is consistent with the view that the Rb-1 gene product plays a general role in the maturation of precursor cells. In contrast, mice heterozygous for a null allele of p53 are predisposed to a spectrum of tumours, while the corresponding homozygotes are viable but show a very high tumour incidence. Thymocytes from p53 homozygotes, unlike wild-type thymocytes, do not show increased levels of apoptosis following treatment with DNA-damaging agents, while response to its induction by other agents is unaltered. Similarly, epithelial cells from the crypts of both small and large intestine of p53-deficient mice are resistant to the induction of apoptosis by γ-irradiation. In contrast, two other early responses of wild-type crypts to γ-irradiation, namely the G2 block and the reduction in bromodeoxyuridine incorporation, are both largely intact in p53-deficient mice. These observations are consistent with the view that p53 is responsible for monitoring DNA damage so that damaged cells can be either repaired or eliminated prior to division.

Tumour suppressor gene mutations in humans and mice: parallels and contrasts.

Tumour suppressor genes prevent cancer development. They can be identified by studying humans, but a full understanding of the mechanisms of their action requires the production of animal models. Mice with mutations in tumour suppressor genes can be produced by gene targeting. The phenotypic consequences of tumour suppressor gene mutations in mice and humans show parallels and contrasts, and both can contribute to the elucidation of disease processes.

Factors influencing the differentiation of embryonal carcinoma and embryo-derived stem cells.

The effects of aggregation, retinoic acid, and medium conditioned by Buffalo rat liver (BRL) cells, alone and in combination, on the differentiation of PSA4TG12 embryonal carcinoma and E14 embryonal stem cells are reported. The observations indicate that BRL-conditioned medium has more than one effect on the differentiation process, that retinoic acid has at least two effects which operate in different concentration ranges, and that both agents influence the choice of differentiation pathway as well as the extent of differentiation.