One-step generation of a conditional allele in mice using a short artificial intron.

Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology.

Dissecting protein function in vivo: Engineering allelic series in mice using CRISPR-Cas9 technology.

Allelic series are extremely valuable genetic tools to study gene function and identify essential structural features of gene products. In mice, allelic series have been engineered using conventional gene targeting in embryonic stem cells or chemical mutagenesis. While these approaches have provided valuable information about the function of genes, they remain cumbersome.

Overlapping Role of SCYL1 and SCYL3 in Maintaining Motor Neuron Viability.

Members of the SCY1-like (SCYL) family of protein kinases are evolutionarily conserved and ubiquitously expressed proteins characterized by an N-terminal pseudokinase domain, centrally located Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats, and an overall disorganized C-terminal segment. In mammals, three family members encoded by genes , , and have been described. Studies have pointed to a role for SCYL1 and SCYL2 in regulating neuronal function and viability in mice and humans, but little is known about the biological function of SCYL3.

SCYL1 does not regulate REST expression and turnover.

A recent study identified SCYL1 as one of the components of the oncogenic STP axis, which promotes triple-negative breast cancer by regulating degradation of the REST tumor suppressor. Contrary to the findings of that study, herein we show by using 3 distinct genetic approaches that SCYL1 does not regulate REST turnover. Specifically, REST protein levels and turnover were identical in Scyl1+/+ and Scyl1-/- mouse embryonic fibroblasts.

Prox1-Heterozygosis Sensitizes the Pancreas to Oncogenic Kras-Induced Neoplastic Transformation.

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas.

Embryonic Expression and Function of the Ink4d Cyclin D-Dependent Kinase Inhibitor.

Here we report the cloning and functional characterization of the cyclin D-dependent kinase 4 and 6 (Cdk4/6) inhibitory protein Cdkn2d/p19 of (-Ink4d). - is the only family gene highly expressed during development and its transcripts were detected maternally and during neurulation. The -Ink4d protein has 63% identity to mouse and human Cdkn2d/p19 and its function as a negative regulator of cell cycle traverse is evolutionary conserved.

Xenopus transgenics: methods using transposons.

The generation of transgenic animals is an essential tool for many genetic strategies. DNA "cut-and-paste" transposon systems can be used to efficiently modify the Xenopus genome. The DNA transposon substrate, harbored on a circularized plasmid, is co-injected into fertilized Xenopus embryos at the one-cell stage together with mRNA encoding the cognate transposase enzyme.

Forward genetic screens in Xenopus using transposon-mediated insertional mutagenesis.

The class II DNA "cut-and-paste" transposons have been used to efficiently modify the Xenopus genome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supplying the transposase enzyme in trans. We have used two methods to remobilize transposons resident in the frog genome: micro-injection of transposase mRNA at the one-cell stage and expression of the enzyme in the germline from a transgene.

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis.

Background: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization.

Transposon transgenesis in Xenopus.

Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies.

Remobilization of Tol2 transposons in Xenopus tropicalis.

Background: The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.

Transgenesis in Xenopus using the Sleeping Beauty transposon system.

Transposon-based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis, and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon.

CDK9/cyclin complexes modulate endoderm induction by direct interaction with Mix.3/mixer.

Mix-related homeodomain proteins are involved in endoderm formation in the early vertebrate embryo. We used a yeast two-hybrid screen to identify proteins that interact with Mix.3/mixer to regulate endoderm induction.

Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR.

The generation of transgenic lines is vital to many genetic strategies and provides useful reagents for cell labeling and lineage-tracing experiments. Transposon-based systems offer simple, yet robust, platforms for transgenesis in the frog. Here, we provide a protocol for a microinjection-based transposon transgenesis method using a 'natural breeding' strategy for the collection of Xenopus tropicalis embryos.

A flk-1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: an in vivo model for vascular studies.

We have used the Sleeping Beauty (SB) transposable element to generate transgenic Xenopus laevis with expression of green fluorescent protein (GFP) in vascular endothelial cells using the frog flk-1 promoter. This is the first characterization of a SB-generated transgenic Xenopus that has tissue-restricted expression. We demonstrate that the transgene integrated into single genomic loci in two independent founder lines and is transmitted through the germline at the expected Mendelian frequencies.

Tol2 transposon-mediated transgenesis in Xenopus tropicalis.

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens.

Cloning and expression pattern of the Xenopus erythropoietin receptor.

Cytokine signaling plays an important role in the survival and differentiation of vertebrate hematopoietic cells. In red blood cells, erythropoietin is a key component of the differentiation program and maintains the homeostasis of the erythroid compartment. In the adult, anemia stimulates high levels of circulating erythropoietin that drives erythropoiesis to restore normal levels of red blood cells in circulation.

Determination of the minimal domains of Mix.3/Mixer required for endoderm development.

The Mix/Bix family of Pax-like homeodomain transcription factors is expressed early in vertebrate development and play important roles in endoderm and mesoderm formation. Like other Pax-related homeodomain proteins, the Mix/Bix family binds DNA as monomers or dimers and dimerization is mediated by the homeodomain. While the Mix/Bix family shares extensive sequence homology within the DNA-binding homeodomain, ectopic expression of these proteins has profoundly different outcomes.

Expression of Xenopus suppressor of cytokine signaling 3 (xSOCS3) is induced by epithelial wounding.

The suppressor of cytokine signaling (SOCS) family of proteins are intracellular mediators of cytokine signaling. These proteins are induced rapidly by cytokine stimulation and act in a classic negative-feedback loop to attenuate the cellular response to the cytokine signal. In this study, we present the cloning and initial characterization of the Xenopus SOCS3 gene.

Evi-1 expression in Xenopus.

The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes.

BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation.

Morphogen-dependent epidermal-specific transacting factors have not been defined in vertebrates. We demonstrate that a member of the grainyhead transcription factor family, Grainyhead-like 1 (XGrhl1) is essential for ectodermal ontogeny in Xenopus laevis. Expression of this factor is restricted to epidermal cells.