ABSTRACT
We have analyzed the immune system in Stat5-deficient mice. Although Stat5a-/- splenocytes have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b-/- splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained by diminished expression of the IL-2 receptor beta chain (IL-2Rbeta), which is a component of the receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b-/- mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the diminished proliferation, basal as well as IL-2- and IL-15-mediated boosting of NK cytolytic activity was also greatly diminished. These data indicate an essential nonredundant role for Stat5b for potent NK cell-mediated proliferation and cytolytic activity.
Subject(s)
Cytotoxicity, Immunologic , DNA-Binding Proteins/immunology , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , Milk Proteins , Trans-Activators/immunology , Animals , Cell Division/immunology , DNA-Binding Proteins/genetics , Gene Expression Regulation/immunology , Interleukin-15/immunology , Interleukin-2/immunology , Mice , Mice, Knockout , STAT5 Transcription Factor , Trans-Activators/geneticsABSTRACT
The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b-/- mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b-/- mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.
Subject(s)
Body Weight/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation , Liver/physiology , Milk Proteins , Sex Differentiation/genetics , Trans-Activators/genetics , Animals , Female , Humans , Male , Mice , Mice, Knockout , STAT5 Transcription Factor , Signal Transduction/genetics , Tumor Suppressor ProteinsABSTRACT
We have investigated the gene targeting frequency at the hprt locus in a range of embryonic stem cell lines selected for variations in cell cycle parameters. Our results show that targeting frequency varies with cell line by as much as 12-fold between nonisogenic lines and 3-fold between isogenic lines and that a nonisogenic line can support homologous recombination events by up to 21-fold more frequently than an isogenic line. This variation is consistent with both insertion and replacement vectors. These results can be explained by an inverse linear correlation of targeting frequencies with cell doubling times. Additionally, by reducing serum concentration in the culture medium the mean cell doubling time for R1 ES cells can be increased from 11.4 to 15.7 h, with a subsequent 15-fold decrease in gene targeting frequency. This change fits the correlation found for the different nonisogenic cell lines. Our observations have important implications when performing gene targeting experiments and explain some of the variation noted between experiments.
Subject(s)
Cell Cycle , Gene Targeting , Stem Cells/cytology , Animals , Cell Line , Embryo, Mammalian/cytology , Hypoxanthine Phosphoribosyltransferase/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred Strains , Mice, Knockout , Reproducibility of ResultsABSTRACT
We have developed a new method for genomic DNA microextraction using acetone/N,N-dimethylformamide. Combining this with 96-well plate PCR allows rapid analysis of large numbers of genomic DNA samples. One application is the analysis of embryonic stem cells for rare gene targeting events. The combination of 96-well plate tissue culture procedures, DNA microextraction and PCR reduces the delays and risks associated with Southern analysis and avoids the need to freeze large numbers of cell clones. In contrast to previously described rapid genomic extraction procedures, the DNA produced using acetone/N,N-dimethylformamide microextraction is sufficiently pure to support reliable amplification of at least 2.2 kb. The efficacy of the combined microextraction and amplification procedure has been demonstrated by the identification of gene targeting events at the mOR gene locus. In addition, we have also detailed an improved 96-well plate freezing protocol for embryonic stem cells that allows long-term storage at -70 degrees C. The simple nature of the combined procedures described lend themselves to future automation.
Subject(s)
DNA/isolation & purification , Freezing , Gene Targeting/methods , Polymerase Chain Reaction , Stem Cells/chemistry , Acetone , Animals , Base Sequence , Dimethyl Sulfoxide , Dimethylformamide , Embryo, Mammalian , Fetal Blood , Genes, mos , Mice , Molecular Sequence DataABSTRACT
The nucleotide sequence of the murine zinc finger (Zf)-encoding gene, mKr5, was determined to allow sequence contiguity to be established. From this, it was determined that the gene encodes a protein containing 14 Zf of the C2H2 type. Genomic mapping also indicated that there are either two allelic loci or a pseudogene for mKr5 present in the murine genome.
Subject(s)
Alleles , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Restriction MappingABSTRACT
The c-mos proto-oncogene encodes a 37-39K cytoplasmic serine/threonine kinase implicated in the meiotic maturation events during murine spermatogenesis and oogenesis. In Xenopus, ectopic expression of pp39mos can promote both the meiotic maturation of oocytes and also arrest the cleavage of blastomeres. To elucidate the role of pp39mos we have generated homozygous mutant mice by gene targeting in embryonic stem cells. These mice are viable and mutant males are fertile, demonstrating that pp39mos is not essential for spermatogenesis. In contrast, mutant females, have a reduced fertility because of the failure of mature eggs to arrest during meiosis. c-mos-/- oocytes undergo germinal vesicle breakdown and extrusion of both polar bodies followed in some cases by progression into cleavage. Mutant females also develop ovarian cysts. These results demonstrate that a major role for pp39mos is to prevent the spontaneous parthenogenetic activation of unfertilized eggs.
