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Biomedical subjects

R Jaenisch

Publications and source records attributed to R Jaenisch.

At least 235 records · Page 13Linked to original sources
PubMed1983

Retroviruses and mouse embryos: a model system in which to study gene expression in development and differentiation.

Early mouse embryos exposed to Moloney leukaemia virus (M-MuL V) produce substrains of mice, designated Mov-1 to Mov-14, that transmit the virus genetically from one generation to the next. In some substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. The effect of cellular differentiation on virus expression was investigated by introducing M-MuL V into preimplantation or postimplantation mouse embryos, or into embryonal carcinoma cells (EC cells) in tissue culture. Whereas preimplantation embryos or EC cells did not permit virus expression, efficient replication occurred in postimplantation embryos or in differentiated cells. The viral genomes introduced into early embryos were highly methylated and non-infectious when analysed in the adult. In contrast, viral genomes introduced into postimplantation embryos remained unmethylated and were infectious in a transfection assay. Similarly, de novo methylation occurred in undifferentiated EC cells but not in differentiated derivatives. These results demonstrate an efficient de novo methylation activity which appears to be involved in the repression of genes introduced into pluripotent embryonic cells and is not observed in cells of the postimplantation embryo or in differentiated cells growing in culture. Integration of M-MuL V into the germ line can lead to recessive lethal mutations. This has been shown for the Mov-13 substrain, as animals homozygous at the Mov-13 locus die between Days 13 and 14 of embryogenesis. This suggests that viral integration occurred in a chromosomal region that is active during, and crucial for, embryonic development.

Animals↗
PubMed1983

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death.

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.

Animals↗
PubMed1983

Endogenous Moloney leukemia virus in nonviremic Mov substrains of mice carries defects in the proviral genome.

Substrains of mice carrying Moloney murine leukemia virus as a Mendelian gene (Mov locus) have been derived previously. Some of these strains, i.e., Mov-3 and Mov-9, develop viremia, whereas others, i.e., Mov-2, Mov-7, and Mov-10, do not regularly activate virus. We previously have molecularly cloned the respective Mov loci and shown that proviral clones derived from the different viral loci were either infectious (Mov-3, Mov-9) or failed to induce infectious virus (Mov-2, Mov-7, Mov-10) in a transfection assay. To analyze the sites affecting infectivity of the latter clones, complementation assays, in vitro recombinations, and marker rescue experiments were performed. Our results show that the three endogenous Moloney murine leukemia virus clones derived from Mov-2, Mov-7, and Mov-10 carry different mutations in the gag-pol region of the proviral genome. No inhibitory effect of flanking mouse sequences on provirus infectivity was observed.

Animals↗
PubMed1982

De novo methylation and expression of retroviral genomes during mouse embryogenesis.

Retrovirus genomes introduced into mouse zygotes by microinjection of cloned DNA, or into morula stage pre-implantation mouse embryos by infection with Moloney murine leukaemia virus (M-MuLV), became de novo methylated and were blocked in expression. No restriction of virus expression and no de novo methylation were observed when post-implantation mouse embryos were infected with virus. Efficient de novo methylation activity may be an important characteristic of gene regulation in early mouse embryos.

Animals↗
PubMed1982

Infectivity and structure of molecular clones obtained from two genetically transmitted Moloney leukemia proviral genomes.

The Mov-2 and Mov-10 substrains of mice, each carrying Moloney leukemia virus (= M-MuLV) in their germ line at the Mov-2 and Mov-10 locus, respectively, do occasionally at a later age (Mov-2) or not at all (Mov-10) activate infectious virus. The M-MuLV proviruses with flanking mouse sequences corresponding to the Mov-2 and Mov-10 locus, respectively, were molecularly cloned. Restriction enzyme analysis revealed no major deletions or insertions in the proviral genomes of the Mov-2 and Mov-10 locus. Both cloned DNAs induced XC plaques in a transfection assay. The specific infectivity, however, was very low and 3T3 cells transfected with the Mov-2 or Mov-10 clone did not produce infectious virus. Removing part of the 5' cellular sequences from the Mov-10 clone did not increase the infectivity. The results suggest that the M-MuLV integrated at the Mov-2 and Mov-10 locus carry a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of non-infectious particles.

Animals↗
PubMed1982

DNA methylation, retroviruses, and embryogenesis.

By exposing preimplantation embryos to Moloney leukemia virus (M-MuLV), we have previously derived substrains of mice designated as Mov-1-Mov-13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M-MuLV was introduced either into preimplantation or post-implantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfectious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.

Animals↗
PubMed1982

De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection of DNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression of proviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.

Animals↗
PubMed1982

Differentiation and virus expression in BALB/Mo mice: endogenous Moloney leukemia virus is not activated in hematopoietic cells.

The endogenous Moloney leukemia virus (M-MuLV) in the BALB/Mo substrain of mice is activated during the first week after birth. Virus replication occurs in cells of the lymphatic system. Lymphoid cells therefore represent the target cells for virus replication and leukemic transformation. To investigate whether virus activation occurs during lymphoid cell differentiation, hematopoietic stem cells carrying the endogenous Mov-1 genome were transplanted to sublethally irradiated BALB/c mice. Effective colonization of the recipients was demonstrated by Southern DNA hybridization. No activation of the endogenous Mov-1 genome occurred during a 4-month observation period after the transplantation. Thus the first step in development of disease in BALB/Mo mice involves activation of the Mov-1 locus in a nonhematopoietic cell followed by superinfection of lymphatic cells and subsequent virus replication and virus spread.

Animals↗
PubMed1982

Cloning of two genetically transmitted Moloney leukemia proviral genomes: correlation between biological activity of the cloned DNA and viral genome activation in the animal.

The Mov-7 and Mov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the Mov-7 locus and Mov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the Mov-9 locus but is not activated in animals carrying the Mov-7 locus. Consequently, only Mov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the Mov-7 and Mov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the Mov-7 and Mov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10(-5) XC plaques per genome equivalent were induced by the Mov-9 clone, whereas only 10(-9) XC plaques per genome equivalent were induced by the Mov-7 clone. Moreover, NIH 3T3 cells transfected with the Mov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with the Mov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at the Mov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in Mov-7 and Mov-9 mice correlates with the biological properties of the respective clones. Genomic DNA from Mov-9 mice was not infectious in the transfection assay (specific infectivity < 10(-7) PFU per genome equivalent). As the only difference between the genomic and the cloned Mov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.

Animals↗
PubMed1981

Infectivity and methylation of retroviral genomes is correlated with expression in the animal.

We studied mechanisms controlling gene expression during animal development using retroviruses as model genes. For this, substrains of mice have been previously derived carrying the Moloney leukemia virus (M-MuLV) in their germ line. Virus activation occurs in some of these substrains at different stages of development, resulting in two classes of viral genomes. The genetically transmitted (endogenous) copy is present in every cell, whereas somatically acquired ("exogenous") copies are carried only in cells that were superinfected. We compared these two classes of M-MuLV genomes using two parameters. DNA sequences of the endogenous M-MuLV genome in all mouse substrains were highly methylated in GCGC, the recognition sequence of the restriction enzyme Hha I, and were not infectious (specific infectivity less than 10(-7) pfu per proviral genome) in a DNA transfection assay. In contrast, the "exogenous" copies were hypomethylated and infectious. These parameters are strongly correlated to genome activity in the animal: only tissues carrying exogenous copies express virus-specific RNA. With the assumption that gene expression of transfected DNA is controlled by mechanisms that are relevant for gene expression in the animal, our results suggest that DNA methylation plays a causative role in gene regulation during development and differentiation.

Animals↗
PubMed1981

Chromosomal position and activation of retroviral genomes inserted into the germ line of mice.

The exogenous Moloney leukemia virus (M-MuLV) was inserted into the germ line of mice by exposing embryos to virus at different stages of embryogenesis. Mice derived from exposed embryos were mosaics with respect to integrated virus. Nine new substrains, designated Mov-5 to Mov-13, were derived, each of which carries a single M-MuLV genome at a different chromosomal position in its germ line. Four substrains, Mov-1 to Mov-4, were derived previously. Restriction enzyme analyses demonstrated that, with the exception of Mov-4 and Mov-6 mice, no major rearrangements or deletions have occurred in the integrated proviral genomes. Infectious virus is not activated in the majority of substrains (Mov-4 to Mov-8 and Mov-10 to Mov-12), whereas the other mice develop viremia. A detailed comparison between Mov-1 and Mov-13 mice demonstrated that the time of virus activation is different. Mov-13 mice activate infectious virus during embryogenesis, leading to a distinct pattern of virus expression in all tissues of the adult, but the viral genome in Mov-1 mice is activated only during the first two weeks after birth, leading to virus expression predominantly in lymphatic organs. Together with previous observations, at least four different phenotypes of virus expression-that is, early virus activation during embryogenesis, virus activation after birth, virus activation late in life and no expression of infectious virus at all-can be distinguished among the 13 substrains. Our results suggest that the chromosomal region at which a viral genome is integrated influences its expression during development and differentiation.

Animals↗
PubMed1981

DNA methylation and gene expression: endogenous retroviral genome becomes infectious after molecular cloning.

The Mov-3 substrain of mice carries Moloney murine leukemia virus as a Mendelian gene in its germ line. All mice segregating the Mov-3 locus activate virus and develop viremia and leukemia. The integrated provirus (i.e., Mov-3 locus) was molecularly cloned from Mov-3 liver DNA as a 16.8 kilobase long EcoRI fragment. Comparison of the cloned and genomic Mov-3 specific EcoRI fragment by restriction enzyme analysis showed no differences in the size of the fragments, indicating that no major sequence rearrangements occurred during cloning. The genomic and cloned Mov-3 DNAs were compared for methylation and infectivity. Analysis with Hha I showed that the genomic proviral and the flanking mouse sequences were methylated at cytosine residues, in contrast to the cloned Mov-3 locus. The cloned Mov-3 locus, however, was highly infectious in a transfection assay (1 x 10(-3) plaque-forming unit per viral genome) in contrast to the genomic Mov-3 DNA (less than 10(-7) per viral genome). Our results suggest that genes containing 5-methylcytosine are not expressed after transfection into susceptible cells and that removal of the methyl groups by molecular cloning in prokaryotes leads to expression generating infectious proviral DNA. If gene expression of transfected DNA is controlled by mechanisms that are relevant for gene expression in the animal, this suggests that DNA methylation may play a causative role in eukaryotic gene regulation.

5-Methylcytosine↗