Generation of a floxed allele of Smad5 for cre-mediated conditional knockout in the mouse

Smad5 is a member of the Smad family of intracellular mediators of BMP signals and in endothelial cells of TGF-beta signals. We and others previously showed that loss of Smad5 in the mouse results in embryonic lethality (between E9.5-E11.5) due to multiple embryonic and extraembryonic defects. To circumvent the early embryonic lethality and to allow tissue- and time-specific Smad5 inactivation, we created a conditional Smad5 allele in the mouse. Floxed Smad5 (Smad5(flE2,Neo/flE2,Neo)) mice were generated in which both exon2 and the Neo-cassette were flanked by loxP sites. Here we demonstrate that embryos with ubiquitous Cre-mediated deletion of Smad5 (Smad5(flDeltaE2/flDeltaE2)) phenocopy the conventional Smad5 knockout mice. Smad5(flE2/flE2) mice are now available and will be a valuable tool to analyze the role of Smad5 beyond its crucial early embryonic function throughout development and postnatal life.     

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 a Bmp2 conditional null allele

Bone morphogenetic proteins (Bmp's) are known to play many important roles in embryogenesis. In addition, recent data from human genetic studies has revealed that Bmp's also have important functions in maintenance of the adult phenotype and aging. The original Bmp2 germline null allele resulted in lethality at embryonic day 7.0-10.5 due to malformation of the amnion/chorion and cardiac malformations. Because the early embryonic lethality of the Bmp2 germline null allele hinders further investigation into Bmp2 function at later stages, we generated a Bmp2 conditional null allele. Using gene targeting in mouse embryonic stem (ES) cells, we introduced LoxP sites upstream and downstream of Bmp2 exon 3 that encodes the mature peptide. Our results indicate that the Bmp2 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after cre recombinase-induced recombination.     

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.     

Generation of a Mig-6 conditional null allele

Mitogen-inducible gene 6 (Mig-6) is a stress-induced gene that serves as a negative regulator of epidermal growth factor (EGF) signaling and acts as a tumor suppressor. Ablation of Mig-6 results in a significant percentage of embryo lethality as well as abnormalities in multiple tissues. To understand the physiological roles of Mig-6, a conditional null allele, Mig-6(f/f) was generated by introducing LoxP sites that flank exons 2 and 4. The Mig-6(f/f) allele was validated by generating recombined Mig-6(-/-) mice using the Zp3-Cre system. The conditional null allele was confirmed by assaying for Mig-6 gene expression in liver, lung, uterus, and skin. The recombined Mig-6(-/-) mice developed pathological changes, such as degenerative joint diseases and skin hyperplasia similar to the previously reported Mig-6 germline null allele. In addition, these mice also had enlarged uteri with endometrial hyperplasia. In summary, this Mig-6(f/f) mouse is a useful tool for the functional study of the Mig-6 gene in a tissue-specific fashion.     

Generation of an Fgf9 conditional null allele

The fibroblast growth factor (FGF) family consists of 22 widely expressed regulatory polypeptides and controls a broad spectrum of cellular processes. Accumulating data show that FGF9 plays important roles both in embryogenesis and in adult tissue homeostasis. Ablation of Fgf9 alleles leads to lethality at the neonatal stage mainly due to malformations of the lung, as well as causing male-to-female sex reversal. To circumvent the neonatal lethality resulting from disruption of the Fgf9 gene, which hinders further characterization of the role of FGF9 in adult tissue function and homeostasis, we generated an Fgf9 conditional null allele for spatiotemporal- and tissue-specific disruption of Fgf9. Using gene targeting in mouse embryonic stem (ES) cells, we introduced two loxP sites flanking exon 1 in the Fgf9 allele, which encodes 93 amino acid residues at the N-terminal of FGF9. Our results indicate that the Fgf9 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after recombination mediated by the Cre recombinase.     

Generation of an Frs2alpha conditional null allele

The fibroblast growth factor (FGF) signaling family controls a broad spectrum of cellular processes in development and adult tissue homeostasis and function, which is expressed in almost all tissues at all stages. FGF receptor substrate 2 alpha (FRS2alpha) is an adaptor protein that recruits downstream substrates to the FGF receptor (FGFR) tyrosine kinase. Disruption of Frs2alpha gene in mice abrogates activation of the mitogen-activated protein kinase pathway by the FGFR and leads to embryonic lethality at day E7.5 post copulation. To circumvent the embryonic lethality resulting from disruption of the Frs2alpha gene, which hinders further characterization of the role of FRS2alpha in adult tissue function and homeostasis, we generated an Frs2alpha conditional null allele for temporally- and tissue-specific disruption of the Frs2alpha gene. Using gene targeting in mouse embryonic stem cells, we introduced two loxP sites flanking the largest coding exon, exon 5, in the Frs2alpha allele. Our results indicate that the floxed Frs2alpha (Frs2alpha(flox)) allele is a true conditional null allele that encodes wildtype activity and is converted to a null allele after Cre recombinase mediated recombination.     

Generation of a conditional allele for the mouse endothelial nitric oxide synthase gene

Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here, we have generated a floxed eNOS allele in which exons 9–12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre‐deleted alleles displayed truncated eNOS protein, lack of vascular NO production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scarring. These findings demonstrate that the floxed allele is normal and it can be converted to a non‐functional eNOS allele through Cre recombination. This mouse will allow time‐ and cell‐specific eNOS deletion. genesis 50:685–692, 2012. © 2012 Wiley Periodicals, Inc.     

Generation of a conditional null allele of jumonji

The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the alpha-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.     

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 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 a Twist1 conditional null allele in the mouse

Twist1 is the mouse ortholog of TWIST1, the human gene mutated in Saethre-Chotzen syndrome. Previously, a Twist1 null allele was generated by gene targeting in mouse embryonic stem cells. Twist1 heterozygous mice develop polydactyly and a craniofacial phenotype similar to Saethre-Chotzen patients. Mice homozygous for the Twist1 null allele die around embryonic day 11.5 (E11.5) with cranial neural tube closure and vascular defects, hindering in vivo studies of Twist1 function at later stages of development. Here, we report the generation of a Twist1 conditional null allele in mice that functions like a wild-type allele but can be converted to a null allele upon Cre-mediated recombination.