Off-Target Deletion of Conditional Dbc1 Allele in the Foxp3YFP-Cre Mouse Line under Specific Setting.
ABSTRACT: The Cre-LoxP conditional knockout strategy has been used extensively to study gene function in a specific cell-type. In this study, the authors tried to engineer mice in which the Dbc1 gene is conditionally knocked out in Treg cells. Unexpectedly, the conditional Dbc1 allele was completely deleted with a low frequency in some Foxp3YFP-Cre mice harboring floxed Dbc1 allele under specific settings. It was found that the germline recombination of floxed Dbc1 allele, which caused Dbc1 knock out mice, occurred in the male Foxp3YFP-Cre mice harboring floxed Dbc1 allele. Even though the authors documented that Foxp3 is expressed in the testis, the germline recombination was not caused by the germline expression of Cre, which was driven by the Foxp3 promoter. The germline recombination may be caused by the unspecific expression of Cre recombinase in the fetus, in which the floxed Dbc1 allele of some stem cells with development potential to germ cells may be recombined. Additionally, this study found that the floxed Dbc1 allele was recombined in non-T cells of some Foxp3Cre Dbc1fl mice, which need to be characterized. Our results also suggest that using male mice with a low frequency of recombined gene allele can reduce the risk of having full knock out mice.
Project description:The transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated in human cancers. Interestingly, STAT3 also maintains the pluripotency and self-renewal of murine embryonic stem cells, and several tissue stem cell types. To investigate whether STAT3 also maintains the small-intestine crypt stem cell, we conditionally inactivated a Floxed Stat3 allele (Stat3(fl)) in murine small-intestine crypt stem cells. Following Cre recombinase expression, apoptosis increased in Stat3(fl/-) experimental crypts relative to Stat3(wt/-) controls before declining. Control Stat3(wt/-) mice carrying a Flox-STOP LacZ reporter transgene stably expressed LacZ after Cre induction. In contrast, Stat3(fl/-) intestine LacZ expression initially increased modestly, before declining to background levels. Quantitative PCRs revealed a similar transient in recombined Stat3(fl) allele levels. Long-term bromodeoxyuridine labelling directly demonstrated that functional STAT3 is required for +4 to +6 region label-retaining small-intestine stem cell survival. Rapid clearance of recombined Stat3(fl/-) cells involves apoptosis potentially induced by elevated c-Myc in non-recombined cells and involves elevated p53 expression and caspase 3 activation. Intriguingly, Stat3(fl/-) intestine recombination triggered dramatically upregulated polycomb transcriptional repressor Bmi1 - potentially accelerating recombined crypt repopulation. In summary, STAT3 activity is absolutely required for small-intestine crypt stem cell survival at both the +4 to +6 label-retaining and crypt base columnar cell locations.
Project description:A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA(-) blood cells. The loss of GPI-linked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA(-) cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.
Project description:Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.
Project description:Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ~2% of spermatozoa. Thus, different "adipocyte-specific" Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.
Project description:ADAMTS9 is the most conserved member of a large family of secreted metalloproteases having diverse functions. Adamts9 null mice die before gastrulation, precluding investigations of its roles later in embryogenesis, in adult mice or disease models. We therefore generated a floxed Adamts9 allele to bypass embryonic lethality. In this mutant, unidirectional loxP sites flank exons 5-8, which encode the catalytic domain, including the protease active site. Mice homozygous for the floxed allele were viable, lacked an overt phenotype, and were fertile. Conversely, mice homozygous for a germ-line deletion produced from the floxed allele by Cre-lox recombination did not survive past gastrulation. Hemizygosity of the deleted Adamts9 in combination with mutant Adamts20 led to cleft palate and severe white spotting as previously described. Previously, Adamts9 haploinsufficiency combined with either Adamts20 or Adamts5 nullizygosity suggested a cooperative role in interdigital web regression, but the outcome of deletion of Adamts9 alone remained unknown. Here, Adamts9 was conditionally deleted in limb mesoderm using Prx1-Cre mice. Unlike other ADAMTS single knockouts, limb-specific Adamts9 deletion resulted in soft-tissue syndactyly (STS) with 100% penetrance and concurrent deletion of Adamts5 increased the severity of STS. Thus, Adamts9 has both non-redundant and cooperative roles in ensuring interdigital web regression. This new allele will be useful for investigating other biological functions of ADAMTS9.
Project description:TAK1 binding protein 1 (TAB1) binds and induces autophosphorylation of TGF-beta activating kinase (TAK1). TAK1, a mitogen-activated kinase kinase kinase, is involved in several distinct signaling pathways including non-Smad pathways for TGF-beta superfamily members and inflammatory responses caused by cytokines. Conventional disruption of the murine Tab1 gene results in late gestational lethality showing intraventricular septum defects and underdeveloped lung alveoli. To gain a better understanding of the roles of TAB1 in different tissues, at different stages of development, and in pathological conditions, we generated Tab1 floxed mice in which the loxP sites flank Exons 9 and 10 to remove the C-terminal region of TAB1 protein necessary for activation of TAK1. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in the epiblast during early embryogenesis, results in deletion of the gene and protein. These homozygous Cre-recombined null embryos display an identical phenotype to conventional null embryos. This animal model will be useful in revealing distinct roles of TAB1 in different tissues at different stages.
Project description:Glucose Regulated Protein (GRP) 94 and GRP78 are critical molecular chaperones and regulators of signaling. Conditional knockout mouse models have revealed tissue specific requirements for GRP94 and GRP78, including selection for allele retention in specific cell types. Here we report the consequences of mammary-targeted knockout of these GRPs. Our studies revealed that MMTV-Cre, Grp94(f/f) mammary glands, despite GRP94 deficiency, exhibited normal proliferation and ductal morphogenesis. Interestingly, MMTV-Cre, Grp78(f/f) mammary glands displayed only slightly reduced GRP78 protein levels, associating with the retention of the non-recombined Grp78 floxed alleles in isolated mammary epithelial cells and displayed phenotypes comparable to wild-type glands. In contrast, transduction of isolated Grp78(f/f) mammary epithelial stem/progenitor cells with adenovirus expressing GFP and Cre-recombinase was successful in GRP78 ablation, and the GFP sorted cells failed to give rise to repopulated mammary glands in de-epithelialized recipient mice. These studies imply GRP78, but not GRP94, is required for mammary gland development.
Project description:Forkhead box P3 (FOXP3)-positive Treg cells are crucial for maintaining immune homeostasis. FOXP3 cooperates with its binding partners to elicit Treg cells' signature and function, but the molecular mechanisms underlying the modulation of the FOXP3 complex remain unclear. Here we report that Deleted in breast cancer 1 (DBC1) is a key subunit of the FOXP3 complex. We found that DBC1 interacts physically with FOXP3, and depletion of DBC1 attenuates FOXP3 degradation in inflammatory conditions. Treg cells from Dbc1-deficient mice were more resistant to inflammation-mediated abrogation of Foxp3 expression and function and delayed the onset and severity of experimental autoimmune encephalomyelitis and colitis in mice. These findings establish a previously unidentified mechanism regulating FOXP3 stability during inflammation and reveal a pathway for potential therapeutic modulation and intervention in inflammatory diseases.
Project description:Magoh encodes a core component of the exon junction complex (EJC), which binds mRNA and regulates mRNA metabolism. Magoh is highly expressed in proliferative tissues during development. EJC components have been implicated in several developmental disorders including TAR syndrome, Richieri-Costa-Pereira syndrome, and intellectual disability. Existing germline null Magoh mice are embryonic lethal as homozygotes and perinatal lethal as heterozygotes, precluding detailed analysis of embryonic and postnatal functions. Here, we report the generation of a new genetic tool to dissect temporal and tissue-specific roles for Magoh in development and adult homeostasis. This Magoh conditional allele has two loxP sites flanking the second exon. Ubiquitous Cre-mediated deletion of the floxed allele in a heterozygous mouse (Magoh(del/+) ) causes 50% reduction of both Magoh mRNA and protein. Magoh(del/+) mice exhibit both microcephaly and hypopigmentation, thus phenocopying germline haploinsufficient Magoh mice. Using Emx1-Cre, we further show that conditional Magoh deletion in neural progenitors during embryonic development also causes microcephaly. We anticipate this novel conditional allele will be a valuable tool for assessing tissue-specific roles for Magoh in mammalian development and postnatal processes.