15 research outputs found

    Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

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    The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3ā€“4 weeks by using GatewayĀ® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner

    Actbāˆ’/āˆ’ embryos are pale and growth retarded at E10.25.

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    <p>(A) Pictures of freshly dissected embryos at E10.25. Pale and growth retarded <i>Actb<sup>āˆ’/āˆ’</sup></i> embryos show no obvious vascular pattern in the embryo proper (middle) or yolk sac (right) compared to the <i>Actb<sup>+/+</sup></i> littermates. 15Ɨ magnification. (B) LacZ stainings of <i>Actb<sup>+/+</sup></i> and <i>Actb<sup>āˆ’/āˆ’</sup></i> yolk sac at E9.5 indicate reduced vascular branching complexity of <i>Actb<sup>āˆ’/āˆ’</sup></i> yolk sacs. 20Ɨ magnification. (C) Whole mount PECAM-1 immunohistochemistry of <i>Actb<sup>+/+</sup></i> and <i>Actb<sup>āˆ’/āˆ’</sup></i> embryos, processed in parallel, shows less coloring, indicating fewer endothelial cells and red blood cells. 15Ɨ magnification. Embryos were imaged on a Leica MS5 (Leica Microsystems) stereomicroscope. Digital images were acquired using a Leica camera.</p

    Absence of beta-actin during primitive erythropoiesis correlates with reduced Gata2 expression levels in the yolk sac.

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    <p>(A) Relative E8.5 yolk sac mRNA levels measured by qRT-PCR of <i>Gata1</i>, <i>Gata2</i>, Hbb-y, Hbb-bh1, Hba and Hbb. We found a 90% decrease of <i>Gata2</i> expression level in <i>Actbāˆ’/āˆ’</i> embryos versus <i>Actb+/+</i> embryos. (B) Relative E8.5 yolk sac mRNA levels measured by qRT-PCR of <i>Gata2</i> target genes: <i>Runx1</i>, Tal1/Scl, <i>c-kit</i> and <i>EpoR</i>. <i>c-kit</i> and <i>EpoR</i> show 50% reduced expression levels in <i>Actbāˆ’/āˆ’</i> embryos versus <i>Actb+/+</i> embryos. (C) <i>Gata2</i> immunohistochemistry on E9.5 yolk sac sections. <i>Gata2</i> expression was detected only in the endoderm of the yolk sac (white arrows). No difference in <i>Gata2</i> expression could be seen between <i>Actbāˆ’/āˆ’</i> embryos and <i>Actb+/+</i> embryos. 60Ɨ magnification. (D) Relative E8.5 and E10.25 yolk sac mRNA levels measured by qRT-PCR of Vegf. At E10.25, we could demonstrate a 50% increase of Vegf mRNA expression. Error bars represent mean Ā±SEM; *P<.05. Immunofluorescence sections were imaged using an Olympus IX81 confocal microscope with Fluoview FV10 software.</p

    Yolk sac erythropoiesis is impaired in Actbāˆ’/āˆ’ embryos.

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    <p>(A) Primitive erythroid colonies (EryP) from E8.5 yolk sacs measured by methylcellulose assays show a dramatic decrease in colony forming potential in <i>Actb<sup>āˆ’/āˆ’</sup></i> embryos compared to <i>Actb<sup>+/+</sup></i> embryos. Results are given as percentage of <i>Actb<sup>+/+</sup></i> embryos absolute number of colonies (100%). (B) Definitive erythroid colonies (BFU-E, CFU-GM and CFU-GEMM) from E9.75 yolk sacs measured by methylcellulose assays show an 85 to 90% decrease in colony forming potential of <i>Actb<sup>āˆ’/āˆ’</sup></i> embryos versus <i>Actb<sup>+/+</sup></i> embryos. Results are given as percentage of <i>Actb<sup>+/+</sup></i> embryos absolute number of colonies (100%). Bars represent mean Ā±SEM; *P<.05, **P<.01, ***P<.001.</p

    Morphologically normal <i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ embryos show improved Gata2 mRNA levels compared to <i>Actbāˆ’/āˆ’</i> embryos.

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    <p>(A) Relative E10.25 mRNA levels measured by qRT-PCR of <i>Gata1</i>, <i>Gata2</i>, Hbb-y, and Hbb-bh1 in yolk sacs of <i>Actb+/+</i>, <i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ embryos with aberrant morphology and <i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ embryos with normal morphology. (B) Relative E10.25 mRNA levels measured by qRT-PCR of Actg1 and <i>Gata2</i> targets: <i>c-kit</i>, and <i>EpoR</i> in yolk sacs of <i>Actb+/+</i>, <i>Actbāˆ’/āˆ’</i> and the two groups of <i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ embryos. (C) Relative E11.5 mRNA levels measured by qRT-PCR of <i>Gata1</i>, <i>Gata2</i>, Hbb-y, and Hbb-bh1 in yolk sacs of <i>Actb+/+</i>, <i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ embryos with aberrant morphology (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067855#pone-0067855-g006" target="_blank">Figure 6C</a>) and <i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ with normal morphology. White bars ā€Š=ā€Š<i>Actb+/+</i>, dark-grey bars ā€Š=ā€Š<i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ with aberrant morphology, light-grey bars ā€Š=ā€Š<i>Actbāˆ’/āˆ’</i> R26+hGata2EpoR-iCre/+ with normal morphology.</p

    Beta-actin binds to specific regions of the <i>Gata2</i> gene.

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    <p>(A) Immunoprecipitation with the anti beta-actin and anti gamma-actin antibodies followed by qRT-PCR with 9 specific primers for the <i>Gata2</i> promotor region yielded 2 loci of interest: amplicon 3 and 8. Error bars represent mean Ā±SEM; *P<.05, **P<.01. (B) Genomic alignment with multiple species showing that amplicon 3 is partly overlapping with a highly conserved region in the <i>Gata2</i> promotor, especially in mammals. Also the localization of the specific amplicons relative to the mouse <i>Gata2</i> gene is shown. Amplicon8 is located between exons 1 and 2. Figure was made using UCSC genome bioinformatics software (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>).</p

    Actbāˆ’/āˆ’ embryos show reduced number of red blood cells and abnormal blood island morphology in the yolk sac.

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    <p>(A) PECAM-1 immunostained sagittal cardiac and somitic section, showing normal development of the left atrium (a), atrioventricular canal (av), left ventricle (v) and regular development of intersomitic vessels. (B) H&E stained yolk sac sections show disrupted blood island morphology with empty, enlarged cavities in E9.5 <i>Actb<sup>āˆ’/āˆ’</sup></i> embryos. A and B at 20Ɨ magnification. (C) H&E stained yolk sac sections show an almost complete absence of blood islands in E10.25 <i>Actb<sup>āˆ’/āˆ’</sup></i> embryos. 40Ɨ magnification. (D) PECAM-1 immunostained yolk sac section showing disorganized endothelial patterning and almost no red blood cells populating the remaining blood islands at E10.25. C and D at 40Ɨ magnification. Sections were imaged using a SNAP-COOL camera (Roper Scientific) mounted on an Olympus Bx51 microscope (Olympus), with Plan Olympus 20Ɨ/0.40 or 40Ɨ/0.65 lens and RSImage Version 1.9.2 software (Roper Scientific).</p

    Keratinocyte-Specific Ablation of RIPK4 Allows Epidermal Cornification but Impairs Skin Barrier Formation

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    In humans, receptor-interacting protein kinase 4 (RIPK4) mutations can lead to the autosomal recessive Bartsocas-Papas and popliteal pterygium syndromes, which are characterized by severe skin defects, pterygia, as well as clefting. We show here that the epithelial fusions observed in RIPK4 full knockout (KO) mice are E-cadherin dependent, as keratinocyte-specific deletion of E-cadherin in RIPK4 full KO mice rescued the tail-to-body fusion and fusion of oral epithelia. To elucidate RIPK4 function in epidermal differentiation and development, we generated epidermis-specific RIPK4 KO mice (RIPK4 EKO). In contrast to RIPK4 full KO epidermis, RIPK4 EKO epidermis was normally stratified and the outside-in skin barrier in RIPK4 EKO mice was largely intact at the trunk, in contrast to the skin covering the head and the outer end of the extremities. However, RIPK4 EKO mice die shortly after birth due to excessive water loss because of loss of tight junction protein claudin-1 localization at the cell membrane, which results in tight junction leakiness. In contrast, mice with keratinocyte-specific RIPK4 deletion during adult life remain viable. Furthermore, our data indicate that epidermis-specific deletion of RIPK4 results in delayed keratinization and stratum corneum maturation and altered lipid organization and is thus indispensable during embryonic development for the formation of a functional inside-out epidermal barrier
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