Direct removal in the mouse of a floxed neo gene from a three-loxp conditional knockout allele by two novel approaches

genesis ◽  
2001 ◽  
Vol 30 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Xiaoling Xu ◽  
Cuiling Li ◽  
Lisa Garrett-Beal ◽  
Denise Larson ◽  
Anthony Wynshaw-Boris ◽  
...  
Keyword(s):  
Conditional Knockout ◽  
Novel Approaches ◽  
Knockout Allele

Generation of a conditional knockout allele for mammalian Spen protein Mint/SHARP

genesis ◽  
2007 ◽  
Vol 45 (5) ◽  
pp. 300-306 ◽  
Author(s):  
Daisuke Yabe ◽  
Hitoshi Fukuda ◽  
Misayo Aoki ◽  
Shuichi Yamada ◽  
Shinji Takebayashi ◽  
...  
Keyword(s):  
Conditional Knockout ◽  
Knockout Allele

scholarly journals Generation of a conditional knockout allele for the NFAT5 gene in mice

2015 ◽  
Vol 5 ◽  
Author(s):  
Christoph Küper ◽  
Franz-Xaver Beck ◽  
Wolfgang Neuhofer
Keyword(s):  
Conditional Knockout ◽  
Knockout Allele

scholarly journals Multiple neurons encode CrebB dependent appetitive long-term memory in the mushroom body circuit

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yves F Widmer ◽  
Cornelia Fritsch ◽  
Magali M Jungo ◽  
Silvia Almeida ◽  
Boris Egger ◽  
...  
Keyword(s):  
Mushroom Body ◽  
Neural Circuit ◽  
Fruit Fly ◽  
Conditional Knockout ◽  
Long Term Memory ◽  
Temporal Precision ◽  
Regulatory Changes ◽  
Transcriptional Memory ◽  
Knockout Allele

Lasting changes in gene expression are critical for the formation of long-term memories (LTMs), depending on the conserved CrebB transcriptional activator. While requirement of distinct neurons in defined circuits for different learning and memory phases have been studied in detail, only little is known regarding the gene regulatory changes that occur within these neurons. We here use the fruit fly as powerful model system to study the neural circuits of CrebB-dependent appetitive olfactory LTM. We edited the CrebB locus to create a GFP-tagged CrebB conditional knockout allele, allowing us to generate mutant, post-mitotic neurons with high spatial and temporal precision. Investigating CrebB-dependence within the mushroom body (MB) circuit we show that MB α/β and α’/β’ neurons as well as MBON α3, but not in dopaminergic neurons require CrebB for LTM. Thus, transcriptional memory traces occur in different neurons within the same neural circuit.

scholarly journals Generation and identification of a conditional knockout allele for the PSMD11 gene in mice

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Linlin Zhao ◽  
Jinming Zhao ◽  
Yingying Zhang ◽  
Lele Wang ◽  
Longyan Zuo ◽  
...  
Keyword(s):  
Embryonic Lethality ◽  
Conditional Knockout ◽  
26S Proteasome ◽  
Whole Body ◽  
Invaluable Tool ◽  
Floxed Allele ◽  
Normal Life Span ◽  
Knockout Allele

Abstract Background Our previous study have shown that the PSMD11 protein was an important survival factor for cancer cells except for its key role in regulation of assembly and activity of the 26S proteasome. To further investigate the role of PSMD11 in carcinogenesis, we constructed a conditional exon 5 floxed allele of PSMD11 (PSMD11flx) in mice. Results It was found that homozygous PSMD11 flx/flx mice showed normal and exhibited a normal life span and fertility, and showed roughly equivalent expression of PSMD11 in various tissues, suggesting that the floxed allele maintained the wild-type function. Cre recombinase could induce efficient knockout of the floxed PSMD11 allele both in vitro and in vivo. Mice with constitutive single allele deletion of PSMD11 derived from intercrossing between PSMD11flx/flx and CMV-Cre mice were all viable and fertile, and showed apparent growth retardation, suggesting that PSMD11 played a significant role in the development of mice pre- or postnatally. No whole-body PSMD11 deficient embryos (PSMD11−/−) were identified in E7.5–8.5 embryos in uteros, indicating that double allele knockout of PSMD11 leads to early embryonic lethality. To avoid embryonic lethality produced by whole-body PSMD11 deletion, we further developed conditional PSMD11 global knockout mice with genotype Flp;FSF-R26CAG − CreERT2/+; PSMD11flx/flx, and demonstrated that PSMD11 could be depleted in a temporal and tissue-specific manner. Meanwhile, it was found that depletion of PSMD11 could induce massive apoptosis in MEFs. Conclusions In summary, our data demonstrated that we have successfully generated a conditional knockout allele of PSMD11 in mice, and found that PSMD11 played a key role in early and postnatal development in mice, the PSMD11 flx/flx mice will be an invaluable tool to explore the functions of PSMD11 in development and diseases.

Genetic Elimination of Prothrombin in Adult Mice Results in Fatal Spontaneous Hemorrhage in the Heart and Central Nervous System

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 395-395
Author(s):  
Eric S. Mullins ◽  
Keith W. Kombrinck ◽  
Kathryn E. Talmage ◽  
Maureen A. Shaw ◽  
David P. Witte ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Conditional Knockout ◽  
Low Grade ◽  
Bleeding Events ◽  
Spontaneous Bleeding ◽  
Term Survival ◽  
Adult Mice ◽  
Hemorrhagic Events ◽  
Knockout Allele

Abstract Thrombin-mediated proteolysis is central to hemostasis, directly controlling both platelet activation and fibrin deposition as well as positively- and negatively-regulating further thrombin generation through the activation of factors XI, VIII, V and protein C. Thrombin also appears to be important in multiple processes distinct from traditional hemostasis, but detailed genetics-based studies have been impeded by the uniform embryonic and perinatal failure of mice with constitutive prothrombin (fII) deficiency. To develop an experimental setting to explore the importance of fII in vascular biology, tissue repair, the inflammatory response and disease processes in adult animals, we have generated mice carrying a conditional prothrombin knockout allele (fIIlox). In the absence of Cre-mediated recombination, homozygous fIIlox/lox mice or compound heterozygous mice carrying one fIIlox allele and one constitutive-null allele were found to carry ~20% and ~10% the normal level of circulating prothrombin, respectively. These fIIlox/lox and fIIlox/− mice exhibited normal developmental and reproductive success, survived well into adulthood and young adults exhibited no appreciable spontaneous bleeding events or other pathologies. However, one-year-old fIIlox/− mice developed modest focal hemosiderin deposits and fibrosis within the heart, consistent with chronic low grade hemorrhage in this tissue. No gross or microscopic pathologies were observed in any other tissue examined in mice carrying an intact conditional knockout allele, regardless of age or gender. The induction of Cre recombinase in adult fIIlox mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fIIlox allele within the liver, loss of hepatic fII mRNA, elimination of detectable circulating prothrombin, and profound derangements in coagulation function. The life-expectancy in adults genetically-depleted of prothrombin was found to be very short (generally 5–7 days), and the loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, most prominently in the heart (100% phenotypic penetrance) and brain (~50% phenotypic penetrance). Microscopic analysis of hearts from mice following deletion of the fII allele revealed widespread hemorrhage within the myocardium, particularly in the subepicardial region, focal ischemia and necrosis, neutrophil infiltrates and early granulation tissue. Gross evidence of blood was observed within the pleural cavity in nearly half of Cre-induced fIIlox/− mice at autopsy, and the lack of appreciable hemorrhage within lung tissue favored the heart as the source of this free blood. Less common hemorrhagic events were observed several other tissues, including skeletal muscle, intestines and testes. Examination of brains from mice lacking fII revealed widespread bleeding in the central nervous system, including dural-based hemorrhage and bleeding into both the brain parenchyma and ventricles. These results imply that a robust hemostatic system is essential to limit spontaneous bleeding events in tissues under repetitive mechanical or pulsatile stress. These findings also demonstrate that low levels of fII are compatible with long-term survival in adult mice, affording the ability to examine fII in a diverse spectrum of disease and physiologic processes. As evidence of this concept, mice lacking fII were challenged with S. aureus peritonitis prior to the onset of hemorrhage. Similar to mice lacking fibrinogen, these animals were found to have a profound deficit in their ability to control this infection, pointing to the need for polymerized fibrin for the effective clearance of this bacteria from the peritoneal cavity.

scholarly journals Putative GTPase GIMAP1 is critical for the development of mature B and T lymphocytes

Blood ◽  
2010 ◽  
Vol 115 (16) ◽  
pp. 3249-3257 ◽  
Author(s):  
Amy Saunders ◽  
Louise M. C. Webb ◽  
Michelle L. Janas ◽  
Amanda Hutchings ◽  
John Pascall ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
T Lymphocyte ◽  
Conditional Knockout ◽  
Lymphocyte Development ◽  
Single Positive ◽  
Mature Lymphocyte ◽  
Knockout Allele ◽  
B And T Lymphocytes

Abstract The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene GIMAP1. Homozygous loss of this allele under the influence of the lymphoid-expressed hCD2-iCre recombinase transgene led to severe (> 85%) deficiency of mature T lymphocytes and, unexpectedly, of mature B lymphocytes. By contrast there was little effect of GIMAP1 deletion on immature lymphocytes in either B or T lineages, although in vitro studies showed a shortening of the survival time of both immature and mature CD4+ single-positive thymocytes. These findings show a vital requirement for GIMAP1 in mature lymphocyte development/survival and draw attention to the nonredundant roles of members of the GIMAP GTPase family in these processes.

scholarly journals Depletion of SMC5/6 sensitizes male germ cells to DNA damage

2018 ◽  
Vol 29 (25) ◽  
pp. 3003-3016 ◽  
Author(s):  
G. Hwang ◽  
D. E. Verver ◽  
M. A. Handel ◽  
G. Hamer ◽  
P. W. Jordan
Keyword(s):  
Dna Damage ◽  
Germ Cells ◽  
Conditional Knockout ◽  
Genome Integrity ◽  
Exogenous Dna ◽  
Meiotic Progression ◽  
Etoposide Treatment ◽  
Structural Maintenance Of Chromosomes ◽  
Mitosis And Meiosis ◽  
Knockout Allele

The structural maintenance of chromosomes complex SMC5/6 is thought to be essential for DNA repair and chromosome segregation during mitosis and meiosis. To determine the requirements of the SMC5/6 complex during mouse spermatogenesis we combined a conditional knockout allele for Smc5, with four germ cell–specific Cre-recombinase transgenes, Ddx4-Cre, Stra8-Cre, Spo11-Cre, and Hspa2-Cre, to mutate Smc5 in spermatogonia, in spermatocytes before meiotic entry, during early meiotic stages, and during midmeiotic stages, respectively. Conditional mutation of Smc5 resulted in destabilization of the SMC5/6 complex. Despite this, we observed only mild defects in spermatogenesis. Mutation of Smc5 mediated by Ddx4-Cre and Stra8-Cre resulted in partial loss of preleptotene spermatocytes; however, spermatogenesis progresses and mice are fertile. Mutation of Smc5 via Spo11-Cre or Hspa2-Cre did not result in detectable defects of spermatogenesis. Upon exposure to gamma irradiation or etoposide treatment, each conditional Smc5 mutant demonstrated an increase in the number of enlarged round spermatids with multiple acrosomes and supernumerary chromosome content. We propose that the SMC5/6 complex is not acutely required for premeiotic DNA replication and meiotic progression during mouse spermatogenesis; however, when germ cells are challenged by exogenous DNA damage, the SMC5/6 complex ensures genome integrity, and thus, fertility.

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