Generation of a conditional null allele of NADPH oxidase activator 1 (NOXA1)

NADPH oxidase complexes are multiprotein assemblies that generate reactive oxygen species in a variety of mammalian tissues. The canonical phagocytic oxidase consists of a heterodimeric, enzymatic core comprised of the transmembrane proteins, CYBB andCYBA and is regulated, in part, by an “organizing” function of NCF1 and an “activating” activity of NCF2. In contexts outside of the phagocyte, these regulatory functions may be encoded not only by NCF1 and NCF2, but also alternatively by their respective paralogues, NOXO1 and NOXA1. To allow tissue‐specific dissection of Noxa1 function in mouse, we have generated an allele of Noxa1 suitable for conditional inactivation. Moreover, by crossing Noxa1 conditional allele carriers to B6.129S4‐Meox2^tm1(Cre)Sor/J mice, we have generated first, Noxa1‐null heterozygotes, and ultimately, Noxa1‐null homozygotes. Through the thoughtful use of tissue‐specific, Cre‐expressing mouse strains, the Noxa1 conditional allele will offer insight into the roles of NOXA1 in the variety of tissues in which it is expressed. genesis 48:568–575, 2010. © 2010 Wiley‐Liss, Inc.     

Generation of mice with a conditional null allele of the Jagged2 gene

The Notch signaling pathway is an evolutionarily‐conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. The Jagged2 (Jag2) gene, which encodes a ligand for Notch pathway receptors, is required for craniofacial, limb, and T cell development. Mice homozygous for a Jag2 null allele die at birth from cleft palate, precluding study of Jag2 function in postnatal and adult mice. We have generated a Jag2 conditional null allele by flanking the first two exons of the Jag2 gene with loxP sites. Cre‐mediated deletion of the Jag2^flox allele generates the Jag2^del2 allele, which behaves genetically as a Jag2 null allele. This Jag2 conditional null allele will enable investigation of Jag2 function in a variety of tissue‐specific contexts. genesis 48:390–393, 2010. © 2010 Wiley‐Liss, Inc.     

Conditional knockout mouse for tissue-specific disruption of the cyclooxygenase-2 (Cox-2) gene

Cyclooxygenase-2 (Cox-2) modulates many normal functions, and appears to play a role in a wide variety of pathophysiologic conditions. Cox-2 gene expression is induced in many different cell types, in response to many distinct stimuli. We generated a conditional knockout mouse in which critical exons of the Cox-2 gene are flanked with loxP sites. Cox-2(flox/flox) mice appear normal and are fertile. Recombination at the loxP sites, loss of Cox-2 protein expression, and prevention of induced PGE2 accumulation are observed in Cox-2(flox/flox) mouse embryo fibroblasts following infection with an adenovirus expressing CRE recombinase. In vivo recombination at the Cox-2(flox) allele was demonstrated in the liver of Cox-2(flox/flox) mice following intravenous injection of adenovirus expressing CRE recombinase. Spatially and temporally restricted elimination of the Cox-2 gene in Cox-2(flox/flox) conditional knockout mice should provide a valuable tool to analyze the cell type-specific role of Cox-2 in many disease models.     

Male germline recombination of a conditional allele by the widely used Dermo1‐cre (Twist2‐cre) transgene

Conditional gene knockout using the Cre/loxP system is instrumental in advancing our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature that recombination mediated by some Cre transgenes can occur in unexpected tissues. Dermo1‐Cre (Twist2‐Cre) has been widely used to target skeletal lineage cells as well as other mesoderm‐derived cells. Here we report that Dermo1‐Cre exhibits spontaneous male germline recombination activity leading to a Cre‐mediated recombination of a floxed Ptk2 (Protein tyrosine kinase 2, also known as Fak [Focal adhesion kinase]) allele but not a floxed Rb1cc1 (RB1 inducible coiled‐coil 1, also known as Fip200 [FAK‐family Interacting Protein of 200 kDa]) allele at high frequency. This ectopic germline activity of Dermo1‐Cre occurred in all or none manner in a given litter. We demonstrated that the occurrence of germline recombination activity of Dermo1‐Cre transgene can be avoided by using female mice as parental Dermo1‐Cre carriers.     

Generation of a conditional null allele for Dmp1 in mouse

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.     

Generation of mice with conditional null allele for GdX/Ubl4A

GdX (also named Ubl4A) is a house-keeping gene located on the X chromosome and encodes a protein harboring an ubiquitin-like domain in human and mouse. Although identified in 1988, the function of GdX remains unknown. To elucidate the role of GdX in vivo, we generated a conditional GdX knockout mouse in which Exon 2 was flanked by two loxP sites. We obtained viable and fertile mice with homozygous GdX(flox/flox) or GdX(flox/Y) allele. Germ-line transmission was confirmed by crossing the mouse bearing conditionally targeted allele with an EIIα-Cre transgenic mouse. GdX was successfully depleted in tissues of EIIα-Cre-GdX-null mice. GdX(-/-) and GdX(-/Y) mice are viable and exhibit normal development compared with wild-type littermates within 6 months during our observation. We also observed that GdX knockout male mice were functionally normal in the reproductive system where Ubl4B was specifically expressed. GdX(flox/flox) and GdX(flox/Y) conditional mice provide a tool for further tissue-specific function analysis of the GdX protein under different conditions.     

Generation of conditional knockout alleles for PDGF-C

PDGF-C is a newly identified member of the platelet-derived growth factor (PDGF) family, which is involved in multiple cellular functions by signaling through PDGF receptor (PDGFR)-alphaalpha and alphabeta dimers. PDGF-C deficiency is perinatal lethal due to the formation of cleft palate. To further characterize the cellular function of PDGF-C during both embryonic and postnatal development, we have generated two conditional alleles of the Pdgf-c gene in which two loxP sites flank exon 5. Global Cre-mediated excision of the floxed exon 5 in these alleles resulted in a complete loss of PDGF-C expression and caused embryonic defects identical to those previously described for the PDGF-C null embryos. These conditional alleles will therefore be the important genetic tools for dissecting the spatial and temporal roles of PDGF-C during development and in adult tissues. Furthermore, from this work, we have also described a simple approach for creating mouse conditional alleles in an efficient manner.     

Generation of mice with a conditional allele of the p120 Ras GTPase-activating protein

p120 Ras GTPase-activating protein (RasGAP) encoded by the rasa1 gene in mice is a prototypical member of the RasGAP family of proteins involved in negative-regulation of the p21 Ras proto-oncogene. RasGAP has been implicated in signal transduction through a number of cell surface receptors. In humans, inactivating mutations in the coding region of the RASA1 gene cause capillary malformation arteriovenous malformation. In mice, generalized disruption of the rasa1 gene results in early embryonic lethality associated with defective vasculogenesis and increased apoptosis of neuronal cells. The early lethality in this mouse model precludes its use to further study the importance of RasGAP as a regulator of cell function. Therefore, to circumvent this problem, we have generated a conditional rasa1 knockout mouse. In this mouse, an exon that encodes a part of the RasGAP protein essential for catalytic activity has been flanked by loxP recognition sites. With the use of different constitutive and inducible Cre transgenic mouse lines, we show that deletion of this exon from the rasa1 locus results in effective loss of expression of catalytically-active RasGAP from a variety of adult tissues. The conditional rasa1 mouse will be useful for the analysis of the role of RasGAP in mature cell types.     

Generation of Axin1 conditional mutant mice

Axin1 is a critical negative regulator of the canonical Wnt-signaling pathway. It is a concentration-limiting factor in the β-catenin degradation complex. Axin1 null mutant mouse embryos died at embryonic day 9.5, precluding direct genetic analysis of the roles of Axin1 in many developmental and physiological processes using these mutant mice. In this study, we have generated mice carrying two directly repeated loxP sites flanking the exon 2 region of the Axin1 gene. We show that floxed-allele-carrying mice (Axin1( fx/fx) ) mice appear normal and fertile. Upon crossing the Axin1( fx/fx) mice to the CMV-Cre transgenic mice, the loxP-flanked exon 2 region that encodes the N-terminus and the conserved regulation of G-protein signaling domain was efficiently deleted by Cre-mediated excision in vivo. Moreover, we show that mouse embryos homozygous for the Cre/loxP-mediated deletion of exon 2 of the Axin1 gene display embryonic lethality and developmental defects similar to those reported for Axin1(-/-) mice. Thus, this Axin1(fx/fx) mouse model will be valuable for systematic tissue-specific dissection of the roles of Axin1 in embryonic and postnatal development and diseases.