Cre-loxP Systems Research Articles

Hypothalamic P62 (SQSTM1) regulates energy balance by modulating leptin signaling.

The multifaceted functions of p62 (SQSTM1) are increasingly recognized, but its role in hypothalamic metabolism-associated neurons for energy balance has yet to be elucidated. Single-nucleus RNA sequencing (snRNA-Seq) was performed on hypothalamic tissues from db/db and db/m mice to explore p62 expression. Overexpression and knockout of p62 in hypothalamic POMC neurons were performed via AAV-mediated gene delivery and Cre-loxP systems. Metabolic outcomes were assessed under normal chow (NCD) and high-fat diet (HFD) conditions. The co-immunoprecipitation and luciferase reporter assays were used to investigate the interaction between p62 and STAT3. The snRNA-Seq analysis found that p62 was ubiquitously expressed in hypothalamic neurons, with significantly higher levels in POMC neurons of db/db mice compared to db/m controls. Under NCD or HFD conditions, the absence of p62 in POMC neurons led to increased body weight, decreased energy expenditure and leptin sensitivity, while its overexpression in POMC neurons produced the opposite phenotype. Mechanistically, p62 interacts with STAT3, facilitating its phosphorylation to initiate POMC transcription and amplify leptin sensitivity. This study demonstrated the capacity of p62 to monogenically regulate the obesity phenotype and emphasized its dual role in managing energy homeostasis through direct modulation of STAT3/POMC signaling and amplification of leptin sensitivity.

Genetic lineage tracing identifies adaptive mechanisms of pancreatic islet β cells in various mouse models of diabetes with distinct age of initiation.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet β cells. Cre-LoxP systems were used to generate islet cell type-specific (α, β, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto β-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled β cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of β cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. β cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into β cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of β cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing β cells could also re-trans-differentiate into insulin-producing β cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of β cells. Our findings shed light on how islet β cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.

Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR-Cas9 and Cre-loxP Systems.

Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR-Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi.

A novel recombinant serotype 4 fowl adenovirus expressing fiber-2 protein of duck adenovirus 3.

Recently, the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and duck adenovirus 3 (DAdV-3) were outbroken and widespread, causing substantial economic losses to the duck industry. Therefore, there is an urgent need to generate a recombinant genetic engineering vaccine candidate against both FAdV-4 and DAdV-3. In this study, a novel recombinant FAdV-4 expressing the Fiber-2 protein of DAdV-3, designated as rFAdV-4-Fiber-2/DAdV-3, was generated based on CRISPR/Cas9 and Cre-LoxP systems. Indirect immunofluorescence assay (IFA) and western blot (WB) showed that the Fiber-2 protein of DAdV-3 in rFAdV-4-Fiber-2/DAdV-3 was expressed successfully. Moreover, the growth curve revealed that rFAdV-4-Fiber-2/DAdV-3 replicated efficiently in LMH cells and even showed a stronger replication ability compared to the wild type FAdV-4. The generation of the recombinant rFAdV-4-Fiber-2/DAdV-3 provides a potential vaccine candidate against both FAdV-4 and DAdV-3.

SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division

Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems

Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.

Tetracycline response element driven Cre causes ectopic recombinase activity independent of transactivator element

ObjectiveTamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity. MethodsAdipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity. ResultsTamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass. ConclusionsThese results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.

A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45–UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Quick validation of genetic quality for conditional alleles in mice.

Site‐specific conditional inactivation technologies using Cre‐loxP or Flp‐FRT systems are becoming increasingly important for the elucidation of gene function and disease mechanism in vivo. A large number of gene knockout mouse models carrying complex conditional alleles have been generated by global community efforts and made available for biomedical researchers. The structures of conditional alleles in these mice are becoming increasingly complex and sophisticated, and so the validation of the genetic quality of these alleles is likewise becoming a laborious task for individual researchers. To ensure the reproducibility of conditional experiments, the researcher should confirm that loxP or FRT is integrated at the correct positions in the genome prior to start of the experiments. We report the successful design of universal PCR primers specific to loxP and FRT for the quick validation of conditional floxed and Flrted alleles. The primer set consists of forward and reverse primers complimentary to the loxP or FRT sequences with partial modifications at the 5ʹ end containing 6‐base restriction endonuclease recognition sites. The universal primer set was tested to detect genomic intervals between a pair of cis‐integrated loxP or FRT and was useful for quickly validating various floxed or Flrted alleles in conditional mice.

Application prospect of common viral tracers in study on brain effect of acupuncture

The feasibility and prospect of viral tracers and mediating functional components are explored in study on brain effect of acupuncture. In the paper, proceeding with viral tracers, the viral tracers used to analyze the structure of specific neural circuits are introduced, as well as their mediated probes, optical/chemical genetics techniques, Cre-LoxP systems, etc. The viral tracers and their functional components can not only mark specifically nerve cells or neural circuits, but also interfere with the function of specific types of neurons or nuclei. They solve some disadvantage of traditional nerve tracing method that only describes the morphology of neurons of one brain region and the simple projection among brain regions, and the indirect and non-specific absorption. The viral tracers and their functional components play the important approach to decoding the mechanism on brain effect of acupuncture when introduced in experimental acupuncture so as to provide an in vivo, real-time and intuitive novel method for a further analysis of neurobiological mechanism on brain effect of acupuncture.

Advantages and Limitations of Cre Mouse Lines Used in Skeletal Research.

The Cre-LoxP technology permits gene ablation in specific cell lineages, at chosen differentiation stages of this lineage and in an inducible manner. It has allowed tremendous advances in our understanding of skeleton biology and related pathophysiological mechanisms, through the generation of loss/gain of function or cell tracing experiments based on the creation of an expanding toolbox of transgenic mice expressing the Cre recombinase in skeletal stem cells, chondrocytes, osteoblasts, or osteoclasts. In this chapter, we provide an overview of the different Cre-LoxP systems and Cre mouse lines used in the bone field, we discuss their advantages, limitations, and we outline best practices to interpret results obtained from the use of Cre mice.

A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

The Cre-loxP recombination system is a powerful tool for genetic manipulation. However, there are widely recognized limitations with chemically inducible Cre-loxP systems, and the UV and blue-light induced systems have phototoxicity and minimal capacity for deep tissue penetration. Here, we develop a far-red light-induced split Cre-loxP system (FISC system) based on a bacteriophytochrome optogenetic system and split-Cre recombinase, enabling optogenetical regulation of genome engineering in vivo solely by utilizing a far-red light (FRL). The FISC system exhibits low background and no detectable photocytotoxicity, while offering efficient FRL-induced DNA recombination. Our in vivo studies showcase the strong organ-penetration capacity of FISC system, markedly outperforming two blue-light-based Cre systems for recombination induction in the liver. Demonstrating its strong clinical relevance, we successfully deploy a FISC system using adeno-associated virus (AAV) delivery. Thus, the FISC system expands the optogenetic toolbox for DNA recombination to achieve spatiotemporally controlled, non-invasive genome engineering in living systems.

Triple-cell lineage tracing by a dual reporter on a single allele.

Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre-, Cre-Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.

Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression

Parkinson’s disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (αSyn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study αSyn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different αSyn constructs: αSyn, αSyn coupled to the fluorescence protein Venus (αSyn-Venus), and αSyn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of αSyn upon incubation with DMSO and FeCl3 along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly αSyn-overexpressing cell lines holding high potential for the screening for modulators of αSyn aggregation and αSyn-mediated toxicity.

Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

SummaryIt is highly desirable to specify human developmental principles in an appropriate human model with advanced genetic tools. However, genetically engineering human cells with lineage-tracing systems has not been achieved. Here we introduce strategies to construct lineage-tracing systems in human embryonic stem cells (hESCs). The AAVS1 locus was suitable for the integration of the conditional reporter. The Cre-LoxP and Flp-FRT systems were highly sensitive, which may cause inaccurate lineage labeling in human cells. The recombination sensitivity and tracing fidelity could be finely tuned by modification of the LoxP recombination site. Moreover, tamoxifen-controllable CreERT2-LoxP and FlpERT2-FRT systems showed compelling advantages in tightly tracing human lineages temporally. In proof-of-principle experiments, we traced human PAX6+ neuroectoderm cells and revealed their full neural lineage differentiation potency both in vitro and in vivo. Devising and optimizing of lineage-tracing systems in hESCs will thus set up a solid foundation for human developmental studies.

Skeletal muscle-specific Cre recombinase expression, controlled by the human α-skeletal actin promoter, improves glucose tolerance in mice fed a high-fat diet.

Cre-loxP systems are frequently used in mouse genetics as research tools for studying tissue-specific functions of numerous genes/proteins. However, the expression of Cre recombinase in a tissue-specific manner often produces undesirable changes in mouse biology that can confound data interpretation when using these tools to generate tissue-specific gene knockout mice. Our objective was to characterise the actions of Cre recombinase in skeletal muscle, and we anticipated that skeletal muscle-specific Cre recombinase expression driven by the human α-skeletal actin (HSA) promoter would influence glucose homeostasis. Eight-week-old HSA-Cre expressing mice and their wild-type littermates were fed a low- or high-fat diet for 12weeks. Glucose homeostasis (glucose/insulin tolerance testing) and whole-body energy metabolism (indirect calorimetry) were assessed. We also measured circulating insulin levels and the muscle expression of key regulators of energy metabolism. Whereas tamoxifen-treated HSA-Cre mice fed a low-fat diet exhibited no alterations in glucose homeostasis, we observed marked improvements in glucose tolerance in tamoxifen-treated, but not corn-oil-treated, HSA-Cre mice fed a high-fat diet vs their wild-type littermates. Moreover, Cre dissociation from heat shock protein 90 and translocation to the nucleus was only seen following tamoxifen treatment. These improvements in glucose tolerance were not due to improvements in insulin sensitivity/signalling or enhanced energy metabolism, but appeared to stem from increases in circulating insulin. The intrinsic glycaemia phenotype in the HSA-Cre mouse necessitates the use of HSA-Cre controls, treated with tamoxifen, when using Cre-loxP models to investigate skeletal muscle-specific gene/protein function and glucose homeostasis.

Promoter Specificity and Efficacy in Conditional and Inducible Transgenic Targeting of Lung Macrophages.

Conditional and inducible Cre-loxP systems are used to target gene deletion to specific cell lineages and tissues through promoter-restricted expression of the bacterial DNA recombinase, Cre. Although Cre-loxP systems are widely used to target gene deletion in lung macrophages, limited data are published on the specificity and efficiency of “macrophage targeting” Cre lines. Using R26-stopfl/fl-TdTomato and tetOn-GFP reporter lines, we assessed the specificity and efficiency of four commercially available Cre driver lines that are often considered “macrophage specific.” We evaluated two conditional (Csf1r-Cre and LysM-Cre) and two inducible [CX3CR1-estrogen receptor-Cre (ERCre) and CD68-rtTA] lines. We assessed Cre activation in six resident lung myeloid populations, as well as activation in lung leukocytes, lung epithelial and endothelial cells, peripheral blood leukocytes, and tissue macrophages of the spleen, bone marrow, and peritoneal cavity. Although Csf1r-Cre and LysM-Cre target resident alveolar macrophages (ResAM) and interstitial macrophages (IM) with high efficiency, neither line is specific for macrophages. Csf1r-Cre targets all leukocyte populations, while LysM-Cre targets dendritic cell, neutrophils, monocytes, and a quarter of lung epithelial cells. CX3CR1-ERCre and CD68-rtTA both target IM, but do not target ResAM. Further, although neither line is specific for macrophages, a pulse-wait administration of tamoxifen or doxycycline can be used to significantly improve IM specificity in these inducible lines. In summary, while Cre-loxP remains a powerful tool to study macrophage function, numerous pitfalls exist. Herein, we document strengths and weaknesses of Csf1r-Cre, LysM-Cre, CX3CR1-ERCre, and CD68-rtTA systems for targeting specific macrophage populations in the lungs and provide data that will aid investigators in selecting the proper strain.

The IntXO-PSL Recombination System Is a Key Component of the Second Maintenance System for Bacillus anthracis Plasmid pXO1.

We previously identified three noncontiguous regions on Bacillus anthracis plasmid pXO1 that comprise a system for accurate plasmid partitioning and maintenance. However, deletion of these regions did not decrease retention of certain shortened pXO1 plasmids during vegetative growth. Using two genetic tools developed for DNA manipulation in B. anthracis (the Cre-loxP and Flp-FRT systems), we found two other noncontiguous pXO1 regions that together are sufficient for plasmid stability. This second pXO1 maintenance system includes the tubZ and tubR genes, characteristic of a type III partitioning system, and the IntXO recombinase gene (GBAA_RS29165), encoding a tyrosine recombinase, along with its adjacent 37-bp perfect stem-loop (PSL) target. Insertion of either the tubZ and tubR genes or the IntXO-PSL system into an unstable mini-pXO1 plasmid did not restore plasmid stability. The need for the two components of the second pXO1 maintenance system follows from the sequential roles of IntXO-PSL in generating monomeric circular daughter pXO1 molecules (thereby presumably preventing dimer catastrophe) and of TubZ/TubR in partitioning the monomers during cell division. We show that the IntXO recombinase deletes DNA regions located between two PSL sites in a manner similar to the actions of the Cre-loxP and Flp-FRT systems. Tyrosine recombinases catalyze cutting and joining reactions between short specific DNA sequences. Three types of reactions occur: integration and excision of DNA segments, inversion of DNA segments, and separation of monomeric forms from replicating circular DNA molecules. Here we show that the newly discovered site-specific IntXO-PSL recombinase system that contributes to the maintenance of the B. anthracis plasmid pXO1 can be used for genome engineering in a manner similar to that of the Cre-loxP or Flp-FRT system.

Smooth muscle CaMKIIδ promotes allergen-induced airway hyperresponsiveness and inflammation.

Airway smooth muscle (ASM) is a key target cell in allergen-induced asthma known to contribute to airway hyperresponsiveness (AHR) and chronic airway remodeling. Changes in ASM calcium homeostasis have been shown to contribute to AHR although the mechanisms and Ca(2+) signal effectors are incompletely understood. In the present study, we tested the function of ASM multifunctional protein kinase Ca(2+)/calmodulin-dependent kinase II (CaMKII) isoforms CaMKIIδ and CaMKIIγ in allergen-induced AHR and airway remodeling in vivo. Using a murine model of atopic asthma, we demonstrate that CaMKIIδ protein is upregulated in ASM derived from ovalbumin (OVA)-treated animals compared to controls. A genetic approach to conditionally knock out smooth muscle CaMKIIδ and CaMKIIγ in separate Cre-loxp systems was validated, and using this loss-of-function approach, the function of these CaMKII isoforms was tested in ovalbumin (OVA)-induced airway remodeling and AHR. OVA treatment in control mice had no effect on ASM remodeling in this model of AHR, and CaMKIIδ knockouts had no independent effects on ASM content. However, at 1 day post-final OVA challenge, OVA-induced AHR was eliminated in the CaMKIIδ knockouts. OVA-induced peribronchial inflammation and bronchoalveolar lavage fluid (BALF) levels of the Th2 cytokine IL-13 were significantly decreased in the CaMKIIδ knockouts. Unexpectedly, we found increased peribronchial eosinophils in the smooth muscle CaMKIIδ knockouts compared to control animals at 1 day post-final challenge, suggesting that lack of ASM CaMKIIδ delays the progression of AHR rather than inhibiting it. Indeed, when AHR was determined at 7 days post-final OVA challenge, CaMKIIδ knockouts showed robust AHR while AHR was fully resolved in OVA-challenged control mice. These in vivo studies demonstrate a role for smooth muscle CaMKIIδ in promoting airway inflammation and AHR and suggest a complex signaling role for CaMKIIδ in regulating ASM function. These studies confirm the diverse roles of ASM cells as immune effectors that control AHR and call for further studies into CaMKIIδ-mediated signaling in ASM cells during disease.

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

BackgroundThe pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.ResultsHere, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.ConclusionsThe i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.