Quantification of adipocyte numbers in transgenic mice via the Cre-LoxP recombination sites.
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ABSTRACT:
SUBMITTER: Moser C
PROVIDER: S-EPMC8385114 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Eye phenotypes were investigated in Le-Cre(Tg/-); Pax6(fl/+) mice, which were expected to show tissue-specific reduction of Pax6 in surface ectoderm derivatives. To provide a better comparison with our previous studies of Pax6(+/-) eye phenotypes, hemizygous Le-Cre(Tg/-) and heterozygous Pax6(fl/+)mice were crossed onto the CBA/Ca genetic background. After the Le-Cre transgene had been backcrossed to CBA/Ca for seven generations, significant eye abnormalities occurred in some hemizygous Le-Cre(Tg/-); Pax6(+/+) controls (without a floxed Pax6(fl) allele) as well as experimental Le-Cre(Tg/-); Pax6(fl/+) mice. However, no abnormalities were seen in Le-Cre(-/-); Pax6(fl/+) or Le-Cre(-/-); Pax6(+/+) controls (without the Le-Cre transgene). The severity and frequency of the eye abnormalities in Le-Cre(Tg/-); Pax6(+/+) control mice diminished after backcrossing Le-Cre(Tg/-) mice to the original FVB/N strain for two generations, showing that the effect was reversible. This genetic background effect suggests that the eye abnormalities are a consequence of an interaction between the Le-Cre transgene and alleles of unknown modifier genes present in certain genetic backgrounds. The abnormalities were also ameliorated by introducing additional Pax6 gene copies on a CBA/Ca background, suggesting involvement of Pax6 depletion in Le-Cre(Tg/-); Pax6(+/+) mice rather than direct action of Cre recombinase on cryptic pseudo-loxP sites. One possibility is that expression of Cre recombinase from the Pax6-Le regulatory sequences in the Le-Cre transgene depletes cofactors required for endogenous Pax6 gene expression. Our observation that eye abnormalities can occur in hemizygous Le-Cre(Tg/-); Pax6(+/+) mice, in the absence of a floxed allele, demonstrates the importance of including all the relevant genetic controls in Cre-loxP experiments.
| S-EPMC4182886 | biostudies-literature
Project description:Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER(Tm) and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1(PB)-CreER(Tm);R26R(lacZ) mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the ?-galactosidase (?-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of ?-cells were ?-gal+; in ?-cells placed after 1 week, 46.2±5.0% were ?-gal+; after 2 weeks, 26.3±7.0% were ?-gal+; and after 4 weeks, 1.9±0.9% were ?-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.
| S-EPMC3314663 | biostudies-literature
Project description:The Cre/loxP system is an indispensable tool for temporal and spatial control of gene function in mice. Many mice that express Cre and carry loxP sites in their genomes have been bred for functional analysis of various genes in vivo. Also, several reporter mice have been generated for monitoring of recombination by the Cre/loxP system. We have developed a Cre reporter gene with DsRed1 and Venus that exhibits a strong red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination in experiments using NIH3T3 cells. However, a transgenic mouse introduced with the same reporter gene exhibits a weak red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination. This property manifested ubiquitously in this mouse model and was maintained stably in mouse-derived fibroblasts. Use of the mouse model exhibiting the stronger red fluorescence might result in confusion of the Cre-dependent signal with false signals, because the Venus signal includes some fluorescence in the red region of the spectrum and the DsRed1 signal includes some fluorescence in the green region. However, we fortuitously obtained reporter mice that exhibit a weaker red fluorescence before Cre/loxP-mediated recombination. The use of this mouse model would decrease concern regarding errors in the identification of signals and should increase certainty in the detection of Cre activity in vivo.
| S-EPMC7445058 | biostudies-literature
Project description:Genetically engineered mice are commonly used to model brainstem gliomas in pre-clinical research. One technique for inducing primary tumors in these genetically engineered mice involves delivering viral vectors containing the code for gene-editing proteins. We present a protocol for generating primary brainstem gliomas using the RCAS-TVA retroviral delivery system and the Cre/loxP gene editing system. We describe steps for transfecting and harvesting chicken fibroblast cells, intracranially injecting cells into mice, imaging primary tumors, and treating primary tumors with focal, image-guided brain irradiation. For complete details on the use and execution of this protocol, please refer to Deland et al. (2021).1.
| S-EPMC9950926 | biostudies-literature
Project description:Cre recombinase catalyzes site-specific recombination between two 34-bp loxP sites in a variety of DNA substrates. At the start of the recombination pathway, the loxP sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by Cre-Cre interactions. We describe the structures of synaptic complexes formed between a symmetrized loxP site and two Cre mutants that are defective in strand cleavage. The DNA in these complexes is bent sharply at a single base pair step at one end of the crossover region in a manner that is atypical of protein-induced DNA bends. A large negative roll (-49 degrees) and a positive tilt (16 degrees) open the major groove toward the center of the synapse and compress the minor groove toward the protein-DNA interface. The bend direction of the site appears to determine which of the two DNA substrate strands will be cleaved and exchanged in the initial stages of the recombination pathway. These results provide a structural basis for the observation that exchange of DNA strands proceeds in a defined order in some tyrosine recombinase systems. The Cre-loxS synaptic complex structure supports a model in which synapsis of the loxP sites results in formation of a Holliday junction-like DNA architecture that is maintained through the initial cleavage and strand exchange steps in the site-specific recombination pathway.
| S-EPMC22031 | biostudies-literature
Project description:Cre recombinase residue Arg259 mediates a canonical bidentate hydrogen-bonded contact with Gua27 of its LoxP DNA substrate. Substituting Cyt8-Gua27 with the three other basepairs, to give LoxAT, LoxTA, and LoxGC, reduced Cre-mediated recombination in vitro, with the preference order of Gua27 > Ade27 approximately Thy27 >> Cyt27. While LoxAT and LoxTA exhibited 2.5-fold reduced affinity and 2.5-5-fold slower reaction rates, LoxGC was a barely functional substrate. Its maximum level of turnover was 6-fold reduced over other substrates, and it exhibited 8.5-fold reduced Cre binding and 6.3-fold slower turnover rate. With LoxP, the rate-limiting step for recombination occurs after protein-DNA complex assembly but before completion of the first strand exchange to form the Holliday junction (HJ) intermediate. With the mutant substrates, it occurs after HJ formation. Using an increased DNA-binding E262Q/E266Q "CreQQ" variant, all four substrates react more readily, but with much less difference between them, and maintained the earlier rate-limiting step. The data indicate that Cre discriminates substrates through differences in (i) concentration dependence of active complex assembly, (ii) turnover rate, and (iii) maximum yield of product at saturation, all of which are functions of the Cre-DNA binding interaction. CreQQ suppression of Lox mutant defects implies that coupling between binding and turnover involves a change in Cre subunit DNA affinities during the "conformational switch" that occurs prior to the second strand exchange. These results provide an example of how a DNA-binding enzyme can exert specificity via affinity modulation of conformational transitions that occur along its reaction pathway.
| S-EPMC2891539 | biostudies-literature
Project description:ObjectiveLynch syndrome is caused by germline mutations in DNA mismatch repair genes leading to microsatellite instability (MSI) and colorectal cancer. Mesalazine, commonly used for the treatment of UC, reduces MSI in vitro. Here, we tested natural compounds for such activity and applied mesalazine and thymoquinone in a Msh2(loxP/loxP) Villin-Cre mouse model for Lynch syndrome.DesignFlow cytometry was used for quantitation of mutation rates at a CA13 microsatellite in human colon cancer (HCT116) cells that had been stably transfected with pIREShyg2-enhanced green fluorescent protein/CA13, a reporter for frameshift mutations. Mice were treated for 43 weeks with mesalazine, thymoquinone or control chow. Intestines were analysed for tumour incidence, tumour multiplicity and size. MSI testing was performed from microdissected normal intestinal or tumour tissue, compared with mouse tails and quantified by the number of mutations per marker (NMPM).ResultsBesides mesalazine, thymoquinone significantly improved replication fidelity at 1.25 and 2.5 µM in HCT116 cells. In Msh2(loxP/loxP) Villin-Cre mice, tumour incidence was reduced by mesalazine from 94% to 69% (p=0.04) and to 56% (p=0.003) by thymoquinone. The mean number of tumours was reduced from 3.1 to 1.4 by mesalazine (p=0.004) and to 1.1 by thymoquinone (p<0.001). Interestingly, MSI was reduced in normal intestinal tissue from 1.5 to 1.2 NMPM (p=0.006) and to 1.1 NMPM (p=0.01) by mesalazine and thymoquinone, respectively. Thymoquinone, but not mesalazine, reduced MSI in tumours.ConclusionsMesalazine and thymoquinone reduce tumour incidence and multiplicity in Msh2(loxP/loxP) Villin-Cre mice by reduction of MSI independent of a functional mismatch repair system. Both substances are candidate compounds for chemoprevention in Lynch syndrome mutation carriers.
| S-EPMC4680183 | biostudies-literature
Project description:Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.
| S-EPMC2904746 | biostudies-literature
Project description:Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.
| S-EPMC2803127 | biostudies-literature
Project description:Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.
| S-EPMC3370522 | biostudies-literature