scholarly journals A novel mouse line with epididymal initial segment-specific expression of Cre recombinase driven by the endogenous Lcn9 promoter

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254802
Author(s):  
Qian-qian Gong ◽  
Xiao Wang ◽  
Zhi-lin Dou ◽  
Ke-yi Zhang ◽  
Xiang-guo Liu ◽  
...  
Keyword(s):  
Initial Segment ◽  
Stop Codon ◽  
Dna Recombination ◽  
Cre Recombinase ◽  
Functional Study ◽  
The Novel ◽  
Mouse Line ◽  
Specific Expression ◽  
Reporter Mice

Spermatozoa released from testes undergo a maturation process and acquire the capacity to fertilize ova through epididymal transit. The epididymis is divided into four regions, each with unique morphology, gene profile, luminal microenvironment and distinct function. To study the functions of relevant genes in the epididymal initial segment (IS), a novel IS-specific mouse model, Lcn9-Cre knock-in (KI) mouse line was generated via CRISPR/Cas9 technology. The TAG stop codon was replaced by a 2A-NLS-Cre cassette, resulting in the co-expression of Lcn9 and Cre recombinase. IS-specific Cre expression was first observed from postnatal day 17. Using the Rosa26tdTomato reporter mice, the Cre-mediated DNA recombination was detected exclusively in principal cells. The epididymal IS-specific Cre activity in vivo was further confirmed using Lcn9-Cre mice crossed with a mouse strain carrying Tsc1 floxed alleles (Tsc1flox/+). Cre expression did not affect either normal development or male fecundity. Different from any epididymis-specific Cre mice reported previously, the novel Lcn9-Cre mouse line can be used to introduce entire IS-specific conditional gene editing for gene functional study.

scholarly journals Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Jennifer Doucet ◽  
Hyun Kyung Lee ◽  
Nethangi Udugama ◽  
Jianfeng Xu ◽  
Baoxiu Qi ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Stop Codon ◽  
Phylogenetic Analyses ◽  
Premature Stop Codon ◽  
The Novel ◽  
Specific Expression ◽  
Deletion Mutations ◽  
Brassicaceae Species ◽  
Compatible Pollen ◽  
Compatible Interactions

Abstract Background In the Brassicaceae, the early stages of compatible pollen-stigma interactions are tightly controlled with early checkpoints regulating pollen adhesion, hydration and germination, and pollen tube entry into the stigmatic surface. However, the early signalling events in the stigma which trigger these compatible interactions remain unknown. Results A set of stigma-expressed pseudokinase genes, termed BRASSIKINs (BKNs), were identified and found to be present in only core Brassicaceae genomes. In Arabidopsis thaliana Col-0, BKN1 displayed stigma-specific expression while the BKN2 gene was expressed in other tissues as well. CRISPR deletion mutations were generated for the two tandemly linked BKNs, and very mild hydration defects were observed for wild-type Col-0 pollen when placed on the bkn1/2 mutant stigmas. In further analyses, the predominant transcript for the stigma-specific BKN1 was found to have a premature stop codon in the Col-0 ecotype, but a survey of the 1001 Arabidopsis genomes uncovered three ecotypes that encoded a full-length BKN1 protein. Furthermore, phylogenetic analyses identified intact BKN1 orthologues in the closely related outcrossing Arabidopsis species, A. lyrata and A. halleri. Finally, the BKN pseudokinases were found to be plasma-membrane localized through the dual lipid modification of myristoylation and palmitoylation, and this localization would be consistent with a role in signaling complexes. Conclusion In this study, we have characterized the novel Brassicaceae-specific family of BKN pseudokinase genes, and examined the function of BKN1 and BKN2 in the context of pollen-stigma interactions in A. thaliana Col-0. Additionally, premature stop codons were identified in the predicted stigma specific BKN1 gene in a number of the 1001 A. thaliana ecotype genomes, and this was in contrast to the out-crossing Arabidopsis species which carried intact copies of BKN1. Thus, understanding the function of BKN1 in other Brassicaceae species will be a key direction for future studies.

scholarly journals Live imaging YAP signaling in vivo with fusion reporter mice

2021 ◽  
Author(s):  
Bin Gu ◽  
Brian Bradshaw ◽  
Min Zhu ◽  
Yu Sun ◽  
Sevan Hopyan ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Cell Polarity ◽  
Near Infrared ◽  
Kinase Activity ◽  
Live Imaging ◽  
Mouse Line ◽  
Reporter Mouse ◽  
Reporter Mice ◽  
Mammalian Embryonic Development

YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking Lats kinase activity or blocking cell polarity. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signaling in vivo in many different situations.

Abstract 84: Angiotensin II Type 1a Receptor in the Subfornical Organ is Essential for Blood Pressure Regulation and Hydromineral Effects in Mouse Models of Elevated Brain Renin-Angiotensin System Activity.

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Aline Hilzendeger ◽  
Deborah R Davis ◽  
Martin D Cassell ◽  
Allyn L Mark ◽  
Justin L Grobe ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Sodium Intake ◽  
Dna Recombination ◽  
Renin Angiotensin System ◽  
Cre Recombinase ◽  
Subfornical Organ ◽  
Specific Expression ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Conditional Allele

Elevated brain renin-angiotensin system (RAS) activity is necessary to increase blood pressure in many animal models of hypertension. We tested the hypothesis that AT1A receptors (AT1AR) within the subfornical organ (SFO) are required for the phenotypes that result from an increased brain RAS. We examined the effect of SFO-targeted Cre-recombinase mediated ablation of AT1A in mice treated with DOCA-salt (deoxycorticosterone acetate, 50 mg s.c. + ad lib 0.15 M NaCl). Mice homozygous for a conditional allele of the endogenous AT1A gene (AT1ARflox) were administered an adenovirus encoding Cre-recombinase and eGFP (AdCre), or eGFP alone (AdGFP) into the lateral cerebral ventricle, then treated for 3 weeks with DOCA-salt. AdCre reduced DOCA-salt hypertension (AdGFP baseline: 108±3 mmHg; AdGFP pre-DOCA: 104±3; AdGFP post-DOCA: 136±6 vs AdCre baseline: 116±2; Adcre pre-DOCA: 109±3, Adre post-DOCA: 118±5; P≤0.01), polydipsia (AdGFP+DOCA: 20.6±2.1 mL/day; AdCre+DOCA: 11.6±1.1, P<0.05), and sodium intake (AdGFP+DOCA: 2.6±0.3 mEq/day; AdCre+DOCA: 1.8±0.2, P<0.05). AdCre reduced AT1AR mRNA in the SFO (0.4±0.3 fold of AdGFP), without significant effect in the paraventricular or arcuate nuclei, or cortex; this was paralleled by SFO-specific AT1AR genomic DNA recombination. AdCre also caused SFO-specific recombination in ROSA-TdTomato reporter mice. Complementing the DOCA-salt model, we also examined the effect of AT1AR ablation in the SFO of double-transgenic sRA mice. sRA mice exhibit life-long brain-specific angiotensin overproduction via expression of human angiotensinogen via its own promoter and neuron-specific expression of human renin via the synapsin promoter. In sRA mice bred onto the AT1A conditional genetic background, AdCre significantly attenuated the polydipsia (AdGFP: -0.2±2; AdCre: -9.7±2.6 mL/day) and sodium intake (AdGFP: +0.2±0.7; AdCre: -1.3±0.4 mEq/day). Blood pressure measures are in progress. Together, these data highlight the involvement of SFO AT1A receptors in blood pressure in DOCA-salt model and additionally in hydromineral balance in two different models of increased brain RAS activity.

Thick ascending limb-specific expression of Cre recombinase

2003 ◽  
Vol 285 (1) ◽  
pp. F33-F39 ◽  
Author(s):  
Peter K. Stricklett ◽  
Deborah Taylor ◽  
Raoul D. Nelson ◽  
Donald E. Kohan
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Yellow Fluorescent Protein ◽  
Cre Recombinase ◽  
Specific Gene ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Enhanced Yellow Fluorescent Protein

Evaluation of thick ascending limb (TAL) function has been hindered by the limited ability to selectively examine the function of this nephron segment in vivo. To address this, a Cre/loxP strategy was employed whereby the Tamm-Horsfall (THP) promoter was used to drive Cre recombinase expression in transgenic mice. The THP gene was cloned from a mouse genomic library, and 3.7 kb of the mouse THP 5′-flanking region containing the first noncoding exon of the THP gene were inserted upstream of an epitope-tagged Cre recombinase (THP-CreTag). THP-CreTag transgenic mice were bred with ROSA26-enhanced yellow fluorescent protein (eYFP) mice (contain a loxP-flanked “STOP” sequence 5′ to eYFP), and doubly heterozygous offspring were analyzed. THP and eYFP were expressed in an identical pattern with predominant localization to the renal outer medulla without expression in nonrenal tissues. eYFP did not colocalize with thiazide-sensitive cotransporter (distal tubule) or neuronal nitric oxide synthase (macula densa) expression. THP mRNA expression was detected only in kidney, whereas CreTag mRNA was also present in testes. These data indicate that THP-CreTag transgenic mice can be used for TAL-specific gene recombination in the kidney.

scholarly journals A Cre-amplifier to generate and detect genetic mosaicsin vivo

2019 ◽  
Author(s):  
Francesco Trovato ◽  
Riccardo Parra ◽  
Enrico Pracucci ◽  
Silvia Landi ◽  
Olga Cozzolino ◽  
...  
Keyword(s):  
Gold Standard ◽  
Cre Recombinase ◽  
General Purpose ◽  
Wild Type ◽  
Specific Expression ◽  
Transient Activation ◽  
Powerful Approach

AbstractCre-Lox manipulation is the gold standard for cell-specific expression or knockout of selected genes. However, it is not unusual to deal with conditions of low Cre expression or transient activation, which can often go undetected by conventional Cre-reporters. We designed Beatrix, a general-purpose tool specifically devised to amplify weak Cre recombinase activity, and we used it to develop a powerful approach for thein vivogeneration and detection of sparse mosaics of mutant and wild type cells.

scholarly journals Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca2+/NFATc3 Signaling

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuan Cao ◽  
Hai-ning Wu ◽  
Xiu-li Cao ◽  
Kang-yi Yue ◽  
Wen-juan Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Transmembrane Protein ◽  
Spatial Learning And Memory ◽  
Specific Expression ◽  
Adult Mice ◽  
Molecular Biological Analysis ◽  
Reporter Mice ◽  
Physiological Homeostasis

The quiescence, activation, and subsequent neurogenesis of neural stem cells (NSCs) play essential roles in the physiological homeostasis and pathological repair of the central nervous system. Previous studies indicate that transmembrane protein Ttyh1 is required for the stemness of NSCs, whereas the exact functions in vivo and precise mechanisms are still waiting to be elucidated. By constructing Ttyh1-promoter driven reporter mice, we determined the specific expression of Ttyh1 in quiescent NSCs and niche astrocytes. Further evaluations on Ttyh1 knockout mice revealed that Ttyh1 ablation leads to activated neurogenesis and enhanced spatial learning and memory in adult mice (6–8 weeks). Correspondingly, Ttyh1 deficiency results in accelerated exhaustion of NSC pool and impaired neurogenesis in aged mice (12 months). By RNA-sequencing, bioinformatics and molecular biological analysis, we found that Ttyh1 is involved in the regulation of calcium signaling in NSCs, and transcription factor NFATc3 is a critical effector in quiescence versus cell cycle entry regulated by Ttyh1. Our research uncovered new endogenous mechanisms that regulate quiescence versus activation of NSCs, therefore provide novel targets for the intervention to activate quiescent NSCs to participate in injury repair during pathology and aging.

scholarly journals A novel pathway for MuSK to induce key genes in neuromuscular synapse formation

2003 ◽  
Vol 161 (4) ◽  
pp. 727-736 ◽  
Author(s):  
Eric Lacazette ◽  
Sophie Le Calvez ◽  
Nadesan Gajendran ◽  
Hans Rudolf Brenner
Keyword(s):  
Synapse Formation ◽  
Regulatory Element ◽  
Neuromuscular Synapse ◽  
Expression Vectors ◽  
The Novel ◽  
Reporter Construct ◽  
Specific Expression ◽  
Cultured Myotubes ◽  
Signaling Components

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChRε . We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Smooth muscle expression of Cre recombinase and eGFP in transgenic mice

2002 ◽  
Vol 10 (3) ◽  
pp. 211-215 ◽  
Author(s):  
H.-B. Xin ◽  
K.-Y. Deng ◽  
M. Rishniw ◽  
G. Ji ◽  
M. I. Kotlikoff
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Smooth Muscles ◽  
Cre Recombinase ◽  
Specific Expression

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.

scholarly journals A Tendon-Specific Double Reporter Transgenic Mouse Enables Tracking Cell Lineage and Functions Alteration In Vitro and In Vivo

2021 ◽  
Vol 22 (20) ◽  
pp. 11189
Author(s):  
Rui Chen ◽  
Xunlei Zhou ◽  
Thomas Skutella
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Cell Lineage ◽  
Mouse Line ◽  
Reporter System ◽  
Specific Expression ◽  
Transgenic Mouse Line ◽  
Blue Fluorescent Protein

We generated and characterized a transgenic mouse line with the tendon-specific expression of a double fluorescent reporter system, which will fulfill an unmet need for animal models to support real-time monitoring cell behaviors during tendon development, growth, and repair in vitro and in vivo. The mScarlet red fluorescent protein is driven by the Scleraxis (Scx) promoter to report the cell lineage alteration. The blue fluorescent protein reporter is expressed under the control of the 3.6kb Collagen Type I Alpha 1 Chain (Col1a1) proximal promoter. In this promoter, the existence of two promoter regions named tendon-specific cis-acting elements (TSE1, TSE2) ensure the specific expression of blue fluorescent protein (BFP) in tendon tissue. Collagen I is a crucial marker for tendon regeneration that is a major component of healthy tendons. Thus, the alteration of function during tendon repair can be estimated by BFP expression. After mechanical stimulation, the expression of mScarlet and BFP increased in adipose-derived mesenchymal stem cells (ADMSCs) from our transgenic mouse line, and there was a rising trend on tendon key markers. These results suggest that our tendon-specific double reporter system is a novel model used to study cell re-differentiation and extracellular matrix alteration in vitro and in vivo.

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