A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator.

In an attempt to create an animal model of tissue-specific mitochondrial disease, we generated 'knockout' mice deficient in the heart/muscle isoform of the adenine nucleotide translocator (Ant1). Histological and ultrastructural examination of skeletal muscle from Ant1 null mutants revealed ragged-red muscle fibers and a dramatic proliferation of mitochondria, while examination of the heart revealed cardiac hypertrophy with mitochondrial proliferation. Mitochondria isolated from mutant skeletal muscle exhibited a severe defect in coupled respiration. Ant1 mutant adults also had a resting serum lactate level fourfold higher than that of controls, indicative of metabolic acidosis. Significantly, mutant adults manifested severe exercise intolerance. Therefore, Ant1 mutant mice have the biochemical, histological, metabolic and physiological characteristics of mitochondrial myopathy and cardiomyopathy.

Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6.

In humans, deficiency of the tissue non-specific alkaline phosphatase (TNAP) gene is associated with defective skeletal mineralization. In contrast, mice lacking TNAP generated by homologous recombination using embryonic stem (ES) cells have normal skeletal development. However, at approximately two weeks after birth, homozygous mutant mice develop seizures which are subsequently fatal. Defective metabolism of pyridoxal 5'-phosphate (PLP), characterized by elevated serum PLP levels, results in reduced levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the brain. The mutant seizure phenotype can be rescued by the administration of pyridoxal and a semi-solid diet. Rescued animals subsequently develop defective dentition. This study reveals essential physiological functions of TNAP in the mouse.

Testicular degeneration in Bclw-deficient mice.

To identify genes required for mammalian spermatogenesis, we screened lines of mutant mice created using a retroviral gene-trap system for male infertility. Homozygous ROSA41 male mice exhibit sterility associated with progressive testicular degeneration. Germ-cell defects are first observed at 19 days post-natal (p19). Spermatogenesis is blocked during late spermiogenesis in young adults. Gradual depletion of all stages of germ cells results in a Sertoli-cell-only phenotype by approximately six months of age. Subsequently, almost all Sertoli cells are lost from the seminiferous tubules and the Leydig cell population is reduced. Molecular analysis indicates that the gene mutated is Bclw, a death-protecting member of the Bcl2 family. The mutant allele of Bclw in ROSA41 does not produce a Bclw polypeptide. Expression of Bclw in the testis appears to be restricted to elongating spermatids and Sertoli cells. Potential roles for Bclw in testicular function are discussed.

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

Expression of human adenosine deaminase in murine hematopoietic cells.

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

A systematic molecular genetic approach to study mammalian germline development.

It is difficult to study gene expression in mammalian embryonic germ cells as PGCs constitute only a minor proportion of the mouse embryo. We have overcome this problem by using a novel combination of established molecular and transgenic approaches. A line of mice has been generated in which the cells of the germ lineage express the beta-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages during germline development by use of a stain for beta-gal activity and a fluorescence activated cell sorter. Subsequently, cDNA libraries have been constructed from each germ cell population using a modified lone-linker PCR strategy. These combined cDNA libraries represent genes expressed in PGCs during mammalian germline development. To facilitate a molecular genetic approach to studying mammalian germline development, these cDNA libraries will be pooled to form an arrayed, addressed reference embryonic germ cell cDNA library. In parallel with large-scale cDNA sequencing efforts; genes that are differentially expressed in germ cells will be identified by screening the reference library with probes generated by subtractive hybridization. Complementary DNAs identified using this approach will be analyzed by sequencing, database comparison, genomic mapping and in situ hybridization to ascertain the potential functional importance of each gene to germline development. In addition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function of these genes in this process.

Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia.

Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (-/-) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of approximately 50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at approximately 10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.

Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene.

Oxidative stress resulting from mitochondrially derived reactive oxygen species (ROS) has been hypothesized to damage mitochondrial oxidative phosphorylation (OXPHOS) and to be a factor in aging and degenerative disease. If this hypothesis is correct, then genetically inactivating potential mitochondrial antioxidant enzymes such as glutathione peroxidase-1 (Gpx1; EC 1.11.1.9) should increase mitochondrial ROS production and decrease OXPHOS function. To determine the expression pattern of Gpx1, isoform-specific antibodies were generated and mutant mice were prepared in which the Gpx1 protein was substituted for by beta-galactosidase, driven by the Gpx1 promoter. These experiments revealed that Gpx1 is highly expressed in both the mitochondria and the cytosol of the liver and kidney, but poorly expressed in heart and muscle. To determine the physiological importance of Gpx1, mice lacking Gpx1 were generated by targeted mutagenesis in mouse ES cells. Homozygous mutant Gpx1(tm1Mgr) mice have 20% less body weight than normal animals and increased levels of lipid peroxides in the liver. Moreover, the liver mitochondria were found to release markedly increased hydrogen peroxide, a Gpx1 substrate, and have decreased mitochondrial respiratory control ratio and power output index. Hence, genetic inactivation of Gpx1 resulted in growth retardation, presumably due in part to reduced mitochondrial energy production as a product of increased oxidative stress.

Use of Escherichiu coli (E. coli) lacZ (ß-Galactosidase) as a Reporter Gene.

Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene expression is to place controlling elements (e.g., promoter/enhancer sequences) upstream of a DNA coding sequence, introduce these constructs into transgenic animals or cells in culture, and analyze the levels of gene product produced by the introduced construct. Ideally, such a reporter gene should encode a product that is stable, innocuous to the cell or organism in which it is being expressed, and should be readily detectable, even when present in small quantities.

Analysis of mutations occurring during replication of a SV40 shuttle vector in mammalian cells.

To analyse mutations that arise in mammalian cells we have used a SV40::plasmid shuttle vector containing a portion of the E. coli lacZ gene. We have found that following transfection into monkey Cos-7 cell mutations are not detected in the recovered plasmids at 24 h post transfection, but are found at 48 h post transfection, after the onset of DNA replication. Analysis of the mutant plasmids shows that in almost all cases the mutant phenotype is caused by a deletion or rearrangement of the lacZ gene in the shuttle vector.

An extreme bias in the germ line of XY C57BL/6<->XY FVB/N chimaeric mice.

Chimaeric analysis is a powerful method to address questions about the cell-autonomous nature of defects in spermatogenesis. Symplastic spermatids (sys) mice have a recessive mutation that causes male sterility due to an arrest in germ-cell development during spermiogenesis. Chimaeric mice were generated by aggregation of eight-cell embryos from sys (FVB/N genetic background) and wild-type C57BL/6 (B6) mice to determine whether the male germ-cell defect is cell-autonomous. The resulting FVB/N<->B6 chimaeras (<-> denotes fusion of embryos) were mated with FVB/N mice and coat colour of offspring was used to identify transmission of FVB/N or B6 gametes. Regardless of the relative contribution of B6 to somatic tissues of the chimaeras, almost all (282 of 284; 99.3%) offspring of B6 XY<->XY FVB/N (+/+ or sys/+) males (n = 9) received a FVB/N-derived paternal gamete. After mating of female B6<->FVB/N chimaeras, 51 of 73 (69.9%) offspring received an FVB-derived maternal gamete. Southern blot analysis of different tissues from chimaeric males indicated that, despite the presence of balanced chimaerism in somatic tissues, the germ line in B6 XY<->XY FVB/N mice was essentially FVB/N in composition. Thus there is a strong selective advantage for FVB/N male germ cells over B6 male germ cells in B6<->FVB/N-aggregation chimaeras at some stage during development of the male germ line. Each of three male chimaeras that were either B6 XY<->XY FVB/N (sys/sys) or B6 XX<->XY FVB/N (sys/sys) in composition was sterile, and testis histology was essentially sysmutant. This finding indicates that the function of the gene(s) affected in the sys mutation may be required in the testis, although whether expression is required in germ cells, somatic cells or both remains unknown. The extreme bias in transmission of male gametes has implications for experimental design in studies that use chimaeric analysis to address questions regarding the cell-autonomous nature of germ-cell defects in mice.

Use of a simplified single-site PCR to facilitate cloning of genomic DNA sequences flanking a transgene integration site.

We have used a simplified single-site PCR based strategy to isolate genomic DNA flanking the transgenic insert in a line of transgenic mice. The flanking sequences, which were refractory to more conventional cloning techniques, were isolated and characterized within 1 week. This strategy begins with the annealing and ligation of a single-stranded oligonucleotide to recessed 5' ends of restriction endonu-clease-digested, size-selected transgenic DNA. Following denaturation, a second oligonucleotide is used to prime DNA synthesis from a position within the transgenic sequences to the end of the genomic restriction fragment, finally synthesizing a complement of the ligated oligonucleotide sequence. Subsequently, a PCR is initiated which uses primers specific for the transgenic DNA and the newly synthesized DNA complementary to the ligated oligonucleotide. The only prerequisite data are sequence information for the transgenic DNA and Southern information regarding the size(s) of restriction fragments that contain the flanking genomic material. This report demonstrates one utility of this technique--enabling rapid isolation of specific mammalian genomic DNA sequences.

Stability of a bacterial gene in a bovine papillomavirus-based shuttle vector maintained extrachromosomally in mammalian cells.

In order to analyse the stability of cloned genes in a viral vector we have constructed a shuttle vector based on bovine papillomavirus and the Escherichia coli gene lacZ. Propagation of this vector in mouse C127 cells and analysis of vector sequences in bacteria produced no detectable mutations in the lacZ gene in over 6137 clones analysed. This is 100-fold less than the mutation frequency observed when the same and similar target genes are replicated in monkey COS cells using a simian virus 40-based shuttle vector.

Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells.

Mouse primordial germ cells express tissue non-specific alkaline phosphatase (TNAP) during development, but the widespread expression of another alkaline phosphatase gene in the early embryo limits the potential use of this marker to trace germ cells. To attempt to identify germ cells at all stages during embryonic development and to understand the role of TNAP in germ cell ontogeny, mice carrying a beta geo (lacZ/neor) disrupted allele of the TNAP gene were generated by homologous recombination in embryonic stem cells. Using beta-galactosidase activity, the embryonic pattern of TNAP expression was examined from the blastocyst stage to embryonic day 14. Results indicate that primordial germ cell progenitors do not express TNAP prior to gastrulation although at earlier times TNAP expression is found in an extraembryonic lineage destined to form the chorion. In homozygous mutants, primordial germ cells appear unaffected indicating that TNAP is not essential for their development or migration.

Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene.

An evaluation has been made of the E. coli beta-galactosidase (beta-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-beta-gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of beta-gal under control of this promoter element was observed.

Testis structure in the sys (symplastic spermatids) mouse.

Testes of mice with the recessive insertional mutation termed symplastic spermatids (sys) were assessed for structural and developmental abnormalities. Homozygous (sys/sys) males are infertile due to an abnormality in spermatogenesis leading to azoospermia. The major interruption to spermatogenesis occurs when the intercellular bridges that connect round spermatids open prematurely resulting in the formation of symplasts. Symplasts contain as many as 285 nuclei. Development of spermatids within symplasts is arrested just before, or just after, elongation of the spermatid nuclei begins. Symplasts degenerate and appear to be phagocytized by Sertoli cells and by intratubular macrophages. In addition, degeneration of young round spermatids and also spermatocytes occasionally is observed. Spermatocyte degeneration is substantial in some tubules and leaves them depleted of cells other than basal compartment cells. Sertoli cell abnormalities are prominent and include intracellular vacuolation, absence of apical processes surrounding round spermatids, degeneration, and occasional sloughing. Although reduplication and infolding of the basal lamina is also seen, this does not appear as a common phenomenon. The sys phenotype is first manifest in animals between 19 days and 22 days of age. Considerable variability is seen in testis histology of prepubertal animals; some display degenerating pachytene spermatocytes and virtually no Sertoli cell vacuoles, while others display vacuoles without apparent elevated numbers of degenerating spermatocytes. Although this study has not revealed the primary cell type(s) affected by the insertional inactivation event, it is possible that the abnormalities in the Sertoli cells are responsible for germ cell degeneration as it is generally recognized that deficits in the Sertoli cell can result in major germ cell abnormalities but not vice versa.

Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents.

Structural features of the mouse and rat manchette and the role of the manchette in shaping the spermatid nucleus were investigated. Rod-like elements about 10 nm in diameter and 40-70 nm in length were seen linking the innermost microtubules of the manchette and the outer leaflet of the nuclear envelope in step 8 through step 11 rat and mouse spermatids that either had been routinely fixed for electron microscopy or had been isolated and detergent extracted. Rod-like linkers were also seen joining the nuclear ring to the plasma membrane and nuclear envelope. These linkers may ensure that under normal conditions the manchette remains in a defined position relative to these membranous components. A variety of compounds (taxol, cytoxan, and 5-fluorouracil) were found to perturb the manchette and to affect nuclear shaping. In addition, sys and azh mutant mice were used to determine the consequences of defective manchette formation. These genetic conditions and chemical treatments either produced manchettes that were not in their normal position (azh, sys, and taxol) and/or caused the manchette to appear abnormal (azh, sys, cytoxan, 5-fluorouracil, and taxol), and all resulted in a deformation of the step 9-11 spermatid nucleus. In all instances where the manchette was present, either in normal or ectopic locations, the sectioned nuclear envelope was parallel to the long axis of the microtubules of the manchette. In general, areas of the nuclear envelope where the manchette was not present, or where it was expected to be present but was not, were rounded (normal animals, sys, cytoxan). In addition, there are indications using certain compounds (cytoxan and 5-fluorouracil) as well as in the azh and sys mouse that the manchette may exert pressure to deform the nucleus. It is suggested that the rod-like linkages of the manchette ensure that the nuclear envelope remains at a constant distance from the manchette microtubules and that this is a major factor acting to impart nuclear shape changes on a region of the head caudal to the acrosome during the early elongation phase of spermiogenesis. The manchette microtubules, which are also known to be linked together, may act as a scaffold to deform this part of the nucleus from its spherical shape, perhaps in concert with forces initiated by other structural elements. Evidence from sys animals indicates that structural elements, such as the acrosomal complex over the anterior head (acrosome-actin-nuclear envelope), may affect nuclear shaping over the acrosome-covered portion of the spermatid head.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased affiliative response to vasopressin in mice expressing the V1a receptor from a monogamous vole.

Arginine vasopressin influences male reproductive and social behaviours in several vertebrate taxa through its actions at the V1a receptor in the brain. The neuroanatomical distribution of vasopressin V1a receptors varies greatly between species with different forms of social organization. Here we show that centrally administered arginine vasopressin increases affiliative behaviour in the highly social, monogamous prairie vole, but not in the relatively asocial, promiscuous montane vole. Molecular analyses indicate that gene duplication and/or changes in promoter structure of the prairie vole receptor gene may contribute to the species differences in vasopressin-receptor expression. We further show that mice that are transgenic for the prairie vole receptor gene have a neuroanatomical pattern of receptor binding that is similar to that of the prairie vole, and exhibit increased affiliative behaviour after injection with arginine vasopressin. These data indicate that the pattern of V1a-receptor gene expression in the brain may be functionally associated with species-typical social behaviours in male vertebrates.