A review of current large‐scale mouse knockout efforts

Guan, Chunmei; Ye, Chao; Yang, Xiaomei; Gao, Jiangang

doi:10.1002/dvg.20594

Cited by 121 publications

(81 citation statements)

References 104 publications

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“…Generation of Pannexin 1 Knockout Mice-Pannexin 1-targeted heterozygous C57B1/6N mice were obtained from the Knockout Mouse Project (KOMP) (36), which generates disruption of pannexin 1 by insertion of a strong splice acceptor site between exons 1 and 2 (pannexin1 tm1a(KOMP)Wtsi ). Heterozygous mice were bred to produce homozygous pannexin 1-deficient animals.…”

Section: Reagents-2-phenyl-12-benzisoselenazol-3(2h)-one (Ebselen)mentioning

confidence: 99%

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

Seminario‐Vidal

Okada

Sesma

et al. 2011

Journal of Biological Chemistry

185

212

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ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.The mucociliary clearance process that removes foreign particles and pathogens from the airways is the primary innate defense mechanism in the lung (1). Nucleotides and nucleosides within the airway surface liquid (ASL) 3 regulate key components of mucociliary clearance via activation of epithelial cell surface purinergic receptors (2, 3). ATP activates the G q -coupled P2Y 2 receptor that promotes mucin secretion and ciliary beat frequency and regulates electrolyte transport and ASL volume production by inhibiting sodium absorption (4 -10) and promoting cystic fibrosis transmembrane conductance regulator and Ca 2ϩ -activated Cl Ϫ channel activity (11-16). Adenosine, generated from the hydrolysis of ATP, activates the G scoupled A 2b receptor that promotes cyclic AMP-regulated cystic fibrosis transmembrane conductance regulator Cl Ϫ channel activity (17) and increases cilia beat frequency (5). Although ATP and adenosine are naturally occurring signaling molecules in ASL (18 -22), the mechanisms of airway epithelial ATP release into the ASL are poorly understood.The lung epithelia exhibit a complex cellular composition, and thus, several mechanisms and pathways are likely involved in the release of nucleotides onto airway surfaces. Our recent studies with goblet-like cell models indicate that ATP and other nucleotides are released concomitantly with MUC5AC, a secretory mucin...

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Section: Reagents-2-phenyl-12-benzisoselenazol-3(2h)-one (Ebselen)mentioning

confidence: 99%

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

Seminario‐Vidal

Okada

Sesma

et al. 2011

Journal of Biological Chemistry

185

212

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“…The GlaxoSmithKline P2X7RϪ/Ϫ mice have a lacZ gene inserted at the beginning of the exon 1 region of the P2X7R gene. We also used Pannexin-1-targeted homozygous C57BL/6N mice from KOMP (Guan et al, 2010). Disruption of Panx1 was generated by insertion of a trapping cassette splice acceptor-␤-geo-polyA flanked by Flp-recombinase target sites within intron 2 upstream of the critical exon 3, where it tags the gene with the lacZ reporter.…”

Section: Introductionmentioning

confidence: 99%

Microglia Proliferation Is Controlled by P2X7 Receptors in a Pannexin-1-Independent Manner during Early Embryonic Spinal Cord Invasion

et al. 2012

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Microglia are known to invade the mammalian spinal cord (SC) at an early embryonic stage. While the mechanisms underlying this early colonization of the nervous system are still unknown, we recently found that it is associated, at least partially, with the ability of microglia to proliferate at the onset of motoneuron developmental cell death and of synaptogenesis in mouse embryo (E13.5). In vitro studies have shown that the proliferation and activation of adult microglia can be influenced by the purinergic ionotropic receptor P2X7 via a coupling with Pannexin-1. By performing patch-clamp recordings in situ using a whole-mouse embryonic SC preparation, we show here that embryonic microglia already express functional P2X7R. P2X7R activation evoked a biphasic current in embryonic microglia, which is supposed to reflect large plasma membrane pore opening. However, although embryonic microglia express pannexin-1, this biphasic current was still recorded in microglia of pannexin-1 knock-out embryos, indicating that it rather reflected P2X7R intrinsic pore dilatation. More important, we found that proliferation of embryonic SC microglia, but not their activation state, depends almost entirely on P2X7R by comparing wild-type and P2X7RϪ/Ϫ embryos. Absence of P2X7R led also to a decrease in microglia density. Pannexin-1Ϫ/Ϫ embryos did not exhibit any difference in microglial proliferation, showing that the control of embryonic microglial proliferation by P2X7R does not depend on pannexin-1 expression. These results reveal a developmental role of P2X7R by controlling embryonic SC microglia proliferation at a critical developmental state in the SC of mouse embryos.

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“…The method takes advantage of floxed alleles that are currently available as well as those that are being made through the High Throughput Gene Targeting project 6 . Compared to the use of transgenic expression of Cre, this method provides a rapid way to test gene function in various cell types, as mice carrying the floxed alleles can be used directly for experiments, and the entire virus injection procedure from start to finish can be accomplished in under an hour.…”

Section: Representative Resultsmentioning

confidence: 99%

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Gibson

2011

JoVE

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Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism and schizophrenia 1,2 . Many genes have been studied in the prenatal brain and found crucial to many developmental processes [3][4][5] . However, their function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6 . When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs) 7,8 encoding Cre into the neonatal brain, we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development, including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully in our own lab (unpublished results) and others 8,9 , and can be extended to other viruses, such as lentivirus 9 , as well as to the expression of shRNA or dominant active proteins 10 . Furthermore, by combining this technique with electrophysiology as well as recently-developed optical imaging tools 11 , this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice and rats.

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A review of current large‐scale mouse knockout efforts

Cited by 121 publications

References 104 publications

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

Microglia Proliferation Is Controlled by P2X7 Receptors in a Pannexin-1-Independent Manner during Early Embryonic Spinal Cord Invasion

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

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