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Biomedical subjects

A Bernstein

Publications and source records attributed to A Bernstein.

At least 145 records · Page 8Linked to original sources
PubMed1989

Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology.

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

Animals↗
PubMed1989

High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene.

We have investigated the role of the p53 gene in oncogenesis in vivo by generating transgenic mice carrying murine p53 genomic fragments isolated from a mouse Friend erythroleukemia cell line or BALB/c mouse liver DNA. Elevated levels of p53 mRNA were detected in several tissues of two transgenic lines tested. Increased levels of p53 protein were also detected in most of the tissues analyzed by Western blotting (immunoblotting). Because both transgenes encoded p53 proteins that were antigenically distinct from wild-type p53, it was possible to demonstrate that overexpression of the p53 protein was mostly, if not entirely, due to the expression of the transgenes. Neoplasms developed in 20% of the transgenic mice, with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas. Tissues such as ovaries that expressed the transgene at high levels were not at higher risk of malignant transformation than tissues expressing p53 protein at much lower levels. The long latent period and low penetrance suggest that overexpression of p53 alone is not sufficient to induce malignancies and that additional events are required. These observations provide direct evidence that mutant alleles of the p53 oncogene have oncogenic potential in vivo and that different cell types show intrinsic differences in susceptibility to malignant transformation by p53. Since recent data suggest that p53 may be a recessive oncogene, it is possible that the elevated tumor incidence results from functional inactivation of endogenous p53 by overexpression of the mutant transgene. The high incidence of lung and bone tumors suggests that p53 transgenic mice may provide a useful model to investigate the molecular events that underlie these malignancies in humans.

Alleles↗
PubMed1989

Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage.

Transgenic mice carrying the diphtheria toxin A gene driven by mouse gamma 2-crystallin promoter sequences manifest microphthalmia due to ablation of fiber cells in the ocular lens. Here we map ablation events in the lens by crossing animals hemizygous for the ablation construct with transgenic mice homozygous for the in situ lacZ reporter gene driven by identical gamma 2-crystallin promoter sequences. By comparing the spatial distribution of lacZ-expressing cells and the profile of gamma-crystallin gene expression in the lenses of normal and microphthalmic offspring, the contributions of specific cell types to lens development were examined. The results suggest that phenotypically and developmentally distinct populations of lens fiber cells are able to contribute to the lens nucleus during organogenesis. We also show that dosage of the transgene and its site of integration influence the extent of ablation. In those mice homozygous for the transgene and completely lacking cells of the lens lineage, we show that the sclera, cornea, and ciliary epithelium are reduced in size but, otherwise, reasonably well formed. In contrast, the anterior chamber, iris, and vitreous body are not discernible while the sensory retina is highly convoluted and extensively fills the vitreous chamber.

Animals↗
PubMed1989

Genetic ablation in transgenic mice.

The study of mammalian development has very quickly moved from a largely descriptive endeavour to one in which very precise mechanistic questions can now be formulated and answered. Undoubtedly, advances in this area have been the result of a strong foundation in experimental embryology, the application of molecular genetic techniques to the isolation and analysis of genes of developmental interest, and the ability to manipulate genetically the embryo through transgenic mouse technology. Perhaps the most dramatic illustration of the power of these new technologies is the potential ability to generate mice either that carry mutations in virtually any gene in the germ line through gene targeting in totipotent embryonic stem (ES) cells or that lack specific cell types through the genetic ablation technology reviewed here. Together, these two approaches have made it possible to knock out either a specific gene or a specific cell type in an intact animal and thus offer almost unlimited possibilities for addressing questions concerning the molecular and cellular biology of development. As well, animal models for various human diseases such as dwarfism, immunodeficiencies and demyelination can now be generated. It is clear that further refinements in both gene targeting and genetic ablation technologies are necessary before the full potential of either approach will be realized. Further development of conditional or inducible ablation strategies, coupled with a more precise definition of the cis-acting sequences, responsible for directing gene expression in fully differentiated and more primitive cells, will greatly broaden the range of questions that can be addressed by this approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus.

Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis. c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.

Animals↗
PubMed1988

Patient knowledge of cardio-respiratory drugs.

Eighty-five patients were interviewed to determine their knowledge of their prescribed medicines, the dosing changes they made, the action taken if adverse effects occurred and their information requirements. Most (85%) knew the reason for use of their medicines and many (73%) knew the frequency of use, but few had been told the anticipated duration of use (16%) or been warned of adverse effects (8%). Patients changed the doses of 34% of medicines as they felt appropriate. Forty patients (47%) considered they had experienced side-effects, and 42% of all patients did not disclose their adverse effect. Fifty-eight patients (68%) posed questions about their treatment. These questions concerned efficacy or indication (38%), side-effects (26%), dosing instruction (20%), duration of treatment (18%) and miscellaneous topics (29%).

Adolescent↗
PubMed1988

Genetic tagging of tumor cells with retrovirus vectors: clonal analysis of tumor growth and metastasis in vivo.

Retrovirus vector infection was used to introduce large numbers of unique genetic markers into tumor cell populations for the purpose of analyzing comparative changes in the clonal composition of metastatic versus that of nonmetastatic tumors during their progressive growth in vivo. The cell lines used were SP1, a nonmetastatic, aneuploid mouse mammary adenocarcinoma, and SP1HU9L, a metastatic variant of SP1. Cells were infected with delta e delta pMoTN, a replication-defective retrovirus vector which possesses the dominant selectable neo gene and crippled long terminal repeats. G418r colonies were obtained at a frequency of 4 x 10(-3). Southern blot analysis of a number of clones provided evidence of random and heritable integration of one or two copies of the proviral DNA. Clonal evolution of primary tumor growth and the nature of lineage relationships among spontaneous metastases and primary tumors were analyzed by subcutaneously injecting 10(5) cells from a pooled mixture of 3.6 x 10(2) G418r SP1HU9L or 10(4) G418r SP1 colonies into syngeneic CBA/J mice. The most striking finding was the relative clonal homogeneity of advanced primary tumors; they invariably consisted of a small number (less than 10) of distinct clones despite the fact that hundreds or thousands of uniquely marked clones had been injected. In the case of the metastatic SP1HU9L cells, the nature of these "dominant" clones varied from one tumor to another. Analysis of a number of lung metastases revealed that a proportion of them were derived from dominant primary tumor clones and were composed of one, and sometimes two, distinct progenitors. In some animals, all the lung metastases were derived from a common progenitor clone, whereas in others, each metastatic nodule had a different progenitor. The results show the following. (i) Retrovirus vector infection can be used to introduce large numbers of unique and stable clonal markers into tumor cell populations. (ii) The progeny of a very limited number of clones dominate in advanced primary tumors. (iii) Mammary carcinoma metastases are of mono- or biclonal origin. The significance of the results is discussed.

Adenocarcinoma↗
PubMed1988

Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus.

The p53 gene is rearranged in a high proportion of erythroleukemic cell lines derived from the spleens of mice infected with Friend leukemia virus. These rearrangements result in either the synthesis of a truncated protein or the inactivation of the p53 gene. Here we have molecularly characterized the rearrangements in two murine erythroleukemic cell lines induced by Friend leukemia virus, DP20-1 and CB3, that contain a rearranged p53 gene and fail to express p53 protein. The rearrangement in the DP20-1 cell line is due to the insertion of Friend spleen focus-forming provirus (SFFV) in the 3' end of the p53 gene in intron sequences between exons 9 and 10. Transfection of molecular clones of this SFFV provirus into NIH3T3 cells results in the generation of infectious virus as determined by its ability, in the presence of helper virus, to induce rapid splenomegaly and polycythemia when injected into adult DBA/2J mice. Insertion of SFFV in DP20-1 cells resulted in the expression of an aberrant 2.9 kb RNA species. Analysis of a molecular clone of the rearranged p53 gene in a second cell line, CB3, revealed that the p53 gene in this clone has sustained a large deletion within the p53 gene resulting in the loss of coding sequences between exons 4 and 8. The 5' end of the deletion originates within exon 4 and extends 3' to within the eighth intron. The significance of these findings with regard to the multi-stage nature of Friend virus induced erythroleukemia is discussed.

Animals↗
PubMed1988

Loss of a highly conserved domain on p53 as a result of gene deletion during Friend virus-induced erythroleukemia.

We have investigated a mutation in the p53 gene leading to expression of a truncated 46,000-dalton protein in a Friend virus-induced erythroleukemia cell line. cDNA sequence analysis revealed a deletion of nucleotide sequences in exon 7 and part of exon 8; 17 additional nucleotides, derived from intron 6, were present in the cDNA and served to maintain the reading frame of the encoded protein. Comparison with p53 protein from other species indicated that the region of the molecule missing in p46 included a highly conserved region. In addition, p46 failed to bind SV40 large T antigen in vitro under conditions which promoted binding of p53 to large T. It seems likely, therefore, that an important functional property of p53 may be affected by the mutation.

Amino Acid Sequence↗
PubMed1987

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.

Animals↗
PubMed1987

High efficiency gene transfer and expression in normal murine B lymphocytes.

The introduction of new genetic information into hematopoietic cells offers a new approach for investigating the molecular events controlling differentiation. Retrovirus vectors have been used to transfer new genes with high efficiency into murine hematopoietic cells, primarily of the myeloid lineage. In this report, we show that vectors carrying the dominant, selectable gene for neomycin resistance (neo gene) can successfully infect normal murine B lymphocytes (CFU-B). The infected CFU-B formed colonies in vitro in high concentrations (750 micrograms/ml) of G418, a neomycin analogue. That B lymphocytes contained the neo gene was confirmed by the findings that the putative B cell colonies growing in G418 contained antibody-producing cells and that the cells responding to the B cell mitogen, LPS, were resistant to G418. Infection of normal spleen cells with different vectors containing a variety of transcriptional regulatory sequences resulted in 7-40% of the CFU-B becoming G418 resistant. Introduction of the immunoglobulin heavy chain enhancer into NEO vectors appeared to augment the expression of the neo gene, since the level of G418 resistance was higher in B cells infected with a NEO vector containing the enhancer than in cells infected with a vector lacking the enhancer.

Animals↗
PubMed1987

Modulation of gene expression in multiple hematopoietic cell lineages following retroviral vector gene transfer.

Retrovirus vectors offer a simple and highly efficient method for introducing new genes into mammalian cells. Here, we have examined the efficiency of gene transfer into hematopoietic cells with retrovirus vectors carrying the neomycin (neo) resistance gene expressed from different transcriptional regulatory regions. Direct infection of mouse bone marrow cells resulted in high efficiencies of gene transfer into a variety of myeloid progenitor cells, including pluripotent, erythroid, and granulocyte-macrophage colony-forming cells with all the vectors examined. However, the progeny derived from individual pluripotent progenitor cells infected with different vectors differed markedly in the proportion of G418-resistant progenitor cells, as judged by their ability to survive selection in the drug G418. This biological assay suggests that the highest level of expression was observed when the neo gene was expressed from constructs that contained the herpes thymidine kinase promoter rather than the viral long terminal repeat or the simian virus 40 early region promoter. In contrast, neo gene expression was highest in fibroblasts infected with the vector containing the simian virus 40 early region promoter. These results show that high and sustainable levels of gene expression in hematopoietic cells can be obtained with retrovirus vectors containing appropriate transcriptional regulatory regions.

Animals↗
PubMed1987

Multistage Friend erythroleukemia: independent origin of tumor clones with normal or rearranged p53 cellular oncogenes.

The erythroleukemia induced by Friend virus complex in adult mice is a multistage malignancy characterized by the emergence, late in the disease, of tumorigenic cell clones. We have previously shown that a significant proportion of these clones have unique rearrangements in their cellular p53 oncogene. The clonal relationships among Friend tumor cells isolated in the late stages of Friend erythroleukemia were analyzed by examining the unique integration site of Friend murine leukemia virus and the unique rearrangement in their cellular p53 oncogene. The majority of clones isolated from individual mice infected with Friend virus were clonally related as judged by the site of Friend murine leukemia virus integration. However, Southern gel analysis of DNA from individual Friend cell clones indicated that all of the clones with a normal p53 gene from the same mice were clonally related, but were unrelated to the Friend cell lines with a rearranged p53 gene. These results suggest that Friend tumor cells with rearrangements in their p53 gene arise as the result of a unique transformation event, rather than by progression from already existing tumor cells with a normal p53 gene. They also suggest that such rearrangements in the p53 gene confer a strong selective advantage to these cells in vivo.

Animals↗