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Biomedical subjects

R Jaenisch

Publications and source records attributed to R Jaenisch.

At least 127 records · Page 7Linked to original sources
PubMed1994

Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.

A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation.

Animals↗
PubMed1994

Dependence on p75 for innervation of some sympathetic targets.

The low-affinity neurotrophin receptor p75 binds all neurotrophins with similar affinity. For elucidation of its function, mice bearing a null mutation in the p75 locus were generated. Examination of sympathetic innervation of target tissues revealed that pineal glands lacked innervation and sweat gland innervation was absent or reduced in particular footpads. The absence of adult innervation reflects the failure of axons to reach these targets during development rather than a target deficit. These results indicate that p75 facilitates development of specific populations of sympathetic neurons, for which it may support axon growth.

Adrenergic Fibers↗
PubMed1994

Mice lacking brain-derived neurotrophic factor develop with sensory deficits.

During vertebrate development, neuronal survival depends on target-derived neurotrophic factors. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, can prevent the death of particular peripheral sensory neurons in vitro, and of central motor neurons as well as dopaminergic and cholinergic neurons of the basal forebrain during development. It also prevents the death of motor neurons and midbrain dopaminergic neurons induced by lesions. Here we show that mutant mice lacking BDNF have severe deficiencies in coordination and balance, associated with excessive degeneration in several sensory ganglia including the vestibular ganglion. The few remaining vestibular axons fail to contact the vestibular sensory epithelia, and terminate in the adjacent connective tissue. Survival of sympathetic, midbrain dopaminergic and motor neurons is not affected. These results indicate that BDNF is required for the survival and target innervation of particular neuronal populations.

Animals↗
PubMed1994

Activin/inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction.

Inhibins and activins are dimeric growth factors of the transforming growth factor-beta superfamily, a class of peptides that can regulate the growth and differentiation of a variety of cell types. Recently, activins have been implicated in early vertebrate development through their ability to evoke, in Xenopus embryo explants, both morphological and molecular changes characteristic of mesoderm induction. To understand these processes further, we have used homologous recombination in embryonic stem cells to create mouse strains carrying mutations in the gene encoding the activin/inhibin beta B subunit. These mice are expected to be deficient in activin B (beta B:beta B), activin AB (beta A:beta B), and inhibin B (alpha:beta B). Viable mutant animals were generated, indicating that the beta B subunit is not essential for mesoderm formation in the mouse. Mutant animals suffered, however, from distinct developmental and reproductive defects. An apparent failure of eyelid fusion during late embryonic development led to eye lesions in mutant animals. Whereas beta B-deficient males bred normally, mutant females manifested a profoundly impaired reproductive ability, characterized by perinatal lethality of their offspring. The phenotype of mutant mice suggests that activin beta B (1) plays a role in late fetal development and (2) is critical for female fecundity. In addition, we have found that expression of the related beta A subunit of activin is highly upregulated in ovaries of mutant females. Altered regulation of beta A activin in beta B-deficient mice may contribute to the mutant phenotype.

Activins↗
PubMed1994

DNA methylation and cancer.

Changes in the pattern of DNA methylation have been a consistent finding in cancer cells. The mostly descriptive nature of these studies and the fact that both hypo- and hypermethylation have been observed at various loci have made it difficult to assess whether these changes are causally involved in the transformation process or whether they reflect the altered physiology of rapidly dividing cancer cells. It is clear, however, that DNA methylation plays an important role in the generation of mutations in human tumors. The high incidence of C-to-T transitions found in the p53 tumor-suppressor gene is attributed to the spontaneous deamination of 5-methylcytosine residues. The multiple observations linking DNA methylation to cancer can be resolved in a model proposing that the high rate of mutation at CpG dinucleotides is due in part to methyltransferase-facilitated deamination. Support for a role of DNA methyltransferase as a mutator enzyme is provided by work with a prokaryotic DNA methyltransferase under S-adenosyl-methionine methyl-donor limiting conditions. Methyl-donor limiting conditions might arise in early stages of tumor development, leading to high rates of methyltransferase-mediated CpG mutagenesis, as seen in human tumors. Such a mechanism is consistent with the frequently reported methionine auxotrophy of cancer cells and with the tumorigenic effects of methyl-deficient diets. Methyl deficiency in tumor cells is also consistent with the commonly observed global hypomethylation of tumor cell DNA, despite normal or even high levels of DNA methyltransferase expression.

Animals↗
PubMed1994

Effects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse.

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survival of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (Wv/Wv) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (Wx, Wsh, and W41) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of Wsh/Wx, 25% of Wsh/Wsh, 60% of W41/Wx, and 69.2% of W41/W41 ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W41/Wx, Wsh/Wsh, and W41/W41 mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.

Action Potentials↗
PubMed1994

MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice.

The expression pattern of myogenic regulatory factors and myotome-specific contractile proteins was studied during embryonic development of Myf-5 mutant mice by in situ hybridization and immunohistochemistry. In contrast to somites in wild-type embryos, no expression of myogenin and Myf-6 (MRF4), or any other myotomal markers was detected in mutant animals at E9.0 and E10.0 indicating that Myf-5 plays a crucial role during this developmental period. Significantly, the onset of MyoD expression in rostral somites of E10.5 embryos was unaffected by the Myf-5 mutation suggesting that the activation of the MyoD gene occurs independently of Myf-5 at the correct developmental time. Immediately after the activation of MyoD myogenin transcripts and protein accumulated within the myotome. The first contractile proteins of the sarcomeric apparatus appeared slightly later. By E11.5 the expression of muscle markers were indistinguishable between wild-type and Myf-5 mutant mice. The migration of muscle precursor cells that leave the somites to form limb musculature was monitored in Myf-5-mutant mice by Pax-3 expression. Pax-3-positive cells were equally found in somites and limbs of E10.0 wild-type and mutant mice indicating that myogenic factor expression at the level of somites is not a prerequisite for determination and subsequent migration of limb precursor cells.

Animals↗
PubMed1994

p75-deficient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF.

To understand the role of low-affinity neurotrophin receptor p75 in neural development, we previously generated mice carrying a null mutation in the p75 locus (Lee, K. F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A. H., Chao, M. V. and Jaenisch, R. (1992) Cell 69, 737-749). To elucidate the mechanisms leading to deficits in the peripheral nervous system in p75 mutant mice, we have employed dissociated cultures to examine the responses of p75-deficient dorsal root ganglion (DRG) and superior cervical ganglion (SCG) neurons to different neurotrophins. We found that p75-deficient DRG and SCG neurons displayed a 2- to 3-fold decreased sensitivity to NGF at embryonic day 15 (E15) and postnatal day 3 (P3), respectively, ages that coincide with the peak of naturally occurring cell death. Furthermore, while p75-deficient E15 DRG neurons did not change their response specificity to BDNF, NT-3, and NT-4/5, P3 SCG neurons became more responsive to NT-3 at higher concentrations (nanomolar ranges). These results may help explain the deficits in the peripheral nervous system in p75 mutant mice and provide evidence that p75 can modulate neurotrophin sensitivity in some neurons.

Animals↗
PubMed1994

Beta 2-microglobulin-deficient NOD mice do not develop insulitis or diabetes.

The role of CD8+ T-cells in the development of diabetes in the nonobese diabetic (NOD) mouse remains controversial. Although it is widely agreed that class II-restricted CD4+ T-cells are essential for the development of diabetes in the NOD model, some studies have suggested that CD8+ T-cells are not required for beta-cell destruction. To assess the contribution of CD8+ T-cells to diabetes, we have developed a class of NOD mouse that lacks expression of beta 2-microglobulin (NOD-B2mnull). NOD-B2mnull mice, which lack both class I expression and CD8+ T-cells in the periphery, not only failed to develop diabetes but were completely devoid of insulitis. These results demonstrate an essential role for CD8+ T-cells in the initiation of the autoimmune response to beta-cells in the NOD mouse.

Animals↗
PubMed1993

MyoD or Myf-5 is required for the formation of skeletal muscle.

Mice carrying null mutations in the myogenic regulatory factors Myf-5 or MyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and S1 nuclease analyses indicated that Myf-5(-1-);MyoD(-1-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. Immunohistochemical analysis indicated an absence of desmin-expressing myoblast-like cells. These observations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis.

Animals↗
PubMed1993

Genomic sequence of mouse COL1A1 encoding the collagen propeptides.

The nucleotide sequences of the mouse pro alpha 1(I) gene regions coding for the N- and C-propeptides is reported. The exon-intron structure was highly homologous to human COL1A1 and the deduced amino acid sequences of the N- and C-propeptides showed 67% and 91% identity with the human sequence. This gene sequence information will allow the production of specific gene mutations by site-directed mutagenesis to study the structure and function of these important propeptide domains.

Amino Acid Sequence↗
PubMed1993

Role for DNA methylation in genomic imprinting.

The paternal and maternal genomes are not equivalent and both are required for mammalian development. The difference between the parental genomes is believed to be due to gamete-specific differential modification, a process known as genomic imprinting. The study of transgene methylation has shown that methylation patterns can be inherited in a parent-of-origin-specific manner, suggesting that DNA methylation may play a role in genomic imprinting. The functional significance of DNA methylation in genomic imprinting was strengthened by the recent finding that CpG islands (or sites) in three imprinted genes, H19, insulin-like growth factor 2 (Igf-2), and Igf-2 receptor (Igf-2r), are differentially methylated depending on their parental origin. We have examined the expression of these three imprinted genes in mutant mice that are deficient in DNA methyltransferase activity. We report here that expression of all three genes was affected in mutant embryos: the normally silent paternal allele of the H19 gene was activated, whereas the normally active paternal allele of the Igf-2 gene and the active maternal allele of the Igf-2r gene were repressed. Our results demonstrate that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprinted genes.

Alleles↗
PubMed1993

WT-1 is required for early kidney development.

In humans, germline mutations of the WT-1 tumor suppressor gene are associated with both Wilms' tumors and urogenital malformations. To develop a model system for the molecular analysis of urogenital development, we introduced a mutation into the murine WT-1 tumor suppressor gene by gene targeting in embryonic stem cells. The mutation resulted in embryonic lethality in homozygotes, and examination of mutant embryos revealed a failure of kidney and gonad development. Specifically, at day 11 of gestation, the cells of the metanephric blastema underwent apoptosis, the ureteric bud failed to grow out from the Wolffian duct, and the inductive events that lead to formation of the metanephric kidney did not occur. In addition, the mutation caused abnormal development of the mesothelium, heart, and lungs. Our results establish a crucial role for WT-1 in early urogenital development.

Alkaline Phosphatase↗
PubMed1993

Abrogation of resistance to Theiler's virus-induced demyelination in H-2b mice deficient in beta 2-microglobulin.

Intracerebral infection of susceptible strains of mice with Theiler's virus, a picornavirus, results in central nervous system demyelination, which is similar to multiple sclerosis. Immunogenetic experiments indicate that the MHC (H-2) and, in particular, the D region that controls class I-restricted immune responses, is an important determinant to development of demyelination. We tested whether disruption of beta 2-microglobulin (beta 2-m) would abrogate resistance to demyelinating disease normally observed in H-2b mice. All (C57BI/6 x 129)F3 mice transgenic for homozygous beta 2-m gene disruption (-/-) developed chronic demyelination after Theiler's murine encephalomyelitis virus infection, whereas none of the infected littermates with normal expression of class I MHC (beta 2-m, +/+) developed demyelination. Demyelinated lesions showed class II MHC expression, macrophages, and TNF but no class I MHC expression or CD8+ T cells. No correlation was observed between development of demyelination and delayed-type hypersensitivity responses to virus Ag. Despite the presence of demyelinating lesions, none of the infected beta 2-m (-/-) mice developed neurologic deficits. Infectious virus and virus Ag persisted in the central nervous systems of infected beta 2-m (-/-) mice but not in beta 2-m (+/+) mice. These experiments support the hypothesis that a class I immune response mediated by CD8+ T cells is important in resistance to Theiler's murine encephalomyelitis virus-induced demyelination. Development of chronic neurologic deficits as observed in immunocompetent susceptible strains of mice may be dependent on the presence of class I MHC and CD8+ T cells.

Animals↗
PubMed1993

Mice lacking major histocompatibility complex class I and class II molecules.

Mice lacking major histocompatibility complex (MHC) antigens were generated by mating beta 2-microglobulin-deficient, and therefore class I-deficient, animals with MHC class II-deficient animals. When housed under sterile conditions, the resulting MHC-deficient mice appear healthy, survive for many months, and breed successfully. Phenotypically, MHC-deficient mice are depleted of CD4+ and CD8+ T cells in peripheral lymphoid organs due to a lack of appropriate restricting elements. In contrast, the B-cell compartment of these animals appears intact, and MHC-deficient mice can mount specific antibody responses when challenged with a T-independent antigen. Spleen cells from MHC-deficient animals are poor stimulators and responders in a mixed lymphocyte reaction. Despite their relatively weak cellular immune responses in vitro, MHC-deficient mice reject allogeneic skin grafts with little delay, and grafts from MHC-deficient animals are rapidly rejected by normal allogeneic recipients. Taken together, these results emphasize the plasticity of the immune system and suggest that MHC-deficient mice may be useful for examining compensatory mechanisms in severely immunocompromised animals.

Animals↗
PubMed1993

Germ line transmission of a yeast artificial chromosome spanning the murine alpha 1(I) collagen locus.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed that allows the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protection analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene.

Animals↗
PubMed1993

Normal development and growth of mice carrying a targeted disruption of the alpha 1 retinoic acid receptor gene.

Three unlinked genes encode receptors for retinoic acid (RAR alpha, -beta, and -gamma). Each gene expresses two major protein isoforms differing in the amino terminal A domain by alternative promoter use, fused to common exons encoding most of the receptor protein. The two RAR alpha transcripts (RAR alpha 1 and -alpha 2) are differentially expressed and evolutionarily conserved, as are the RAR beta and -gamma transcripts, suggesting that each isoform may have specific functions in the development of animals. To address the biological function of the alpha 1 receptor, we have disrupted the portion of the RAR alpha gene encoding this isoform by homologous recombination in mouse embryonic stem cells. Surprisingly, offspring homozygous for this mutation were viable and showed no apparently altered phenotype. RNA analysis confirmed that the RAR alpha 1 transcript was absent in homozygous tissues, and no evidence for a compensatory increase of RAR alpha 2 or of another RAR gene was obtained to account for the vitality of the mutant animals. These results clearly demonstrate that loss of RAR alpha 1 function does not disrupt embryonic development and argue for combinatorial or overlapping functions among the RAR isoforms.

Animals↗