Remobilization of Tol2 transposons in Xenopus tropicalis

<p>Abstract</p> <p>Background</p> <p>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 <it>Tol2 </it>element from <it>Oryzias latipes </it>efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, <it>Xenopus tropicalis. Tol2 </it>transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.</p> <p>Results</p> <p>To test whether <it>Tol2 </it>transposons integrated in the <it>Xenopus tropicalis </it>genome are substrates for remobilization, we injected <it>in vitro </it>transcribed <it>Tol2 </it>mRNA into one-cell embryos harbouring a single copy of a <it>Tol2 </it>transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single <it>Tol2 </it>transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the <it>Tol2 </it>transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis.</p> <p>Conclusions</p> <p>In this study, we demonstrate that single copy <it>Tol2 </it>transposons integrated into the <it>Xenopus tropicalis </it>genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.</p

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

<p>Abstract</p> <p>Background</p> <p>The <it>Sleeping Beauty </it>(<it>SB</it>) transposon system has been used for germline transgenesis of the diploid frog, <it>Xenopus tropicalis</it>. Injecting one-cell embryos with plasmid DNA harboring an <it>SB </it>transposon substrate together with mRNA encoding the <it>SB </it>transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that <it>SB </it>transposons stably integrated into the frog genome are effective substrates for remobilization.</p> <p>Results</p> <p>Transgenic frogs that express the <it>SB</it>10 transposase were bred with <it>SB </it>transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the <it>SB </it>transposons. The remobilized <it>SB </it>transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'.</p> <p>Conclusions</p> <p>In this study, we demonstrate that <it>SB </it>transposons integrated into the <it>X. tropicalis </it>genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.</p

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. Similarly, targeted inactivation of SCYL1 in Hek293T cells by using CRIPSR-Cas9 technology did not affect REST steady-state level and turnover. Furthermore, RNA interference-mediated depletion of SCYL1 in Hek293T or MDA-MB-231 cells did not alter REST steady-state level and turnover. Together, our findings indicate that SCYL1 does not contribute to REST turnover and thus do not support a previous study suggesting a role for SCYL1 in mediating REST degradation

REST protein turnover and levels in <i>Scyl1+/+</i> and <i>Scyl1-/-</i> mouse embryonic fibroblasts (MEFs).

<p><b>(</b>A) REST protein turnover in <i>Scyl1+/+</i> and <i>Scyl1-/-</i> MEFs. MEFs were seeded at a density of 250,000 cells per well of 6-well plates. The following day, cells were treated with 10 μg/mL of cycloheximide (CHX) for the indicated times. Endogenous REST, SCYL1, and β-actin levels were determined by western blot analysis. The images are representative of 2 independent experiments performed on 2 independently derived <i>Scyl1+/+</i> and 3 <i>Scyl1-/-</i> MEF lines. Note the absence of SCYL1 in <i>Scyl1-/-</i> MEFs. (B) Quantification of REST protein levels in <i>Scyl1+/+</i> and <i>Scyl1-/-</i> MEFs. The ratio of the band intensity of REST to β-actin was normalized to the ratio of untreated cells (time = 0 h). Data represent mean ± standard error of the mean.</p

Effect of PLK1 inhibition by BI 2536 on REST protein expression and turnover.

<p>(A) Three Hek293T cell lines in which <i>SCYL1</i> was unaltered (Hek293T) and three Hek293T-SCYL1KO cell lines were seeded at a density of 250,000 cells per well of 6-well plates. The following day, the cells were treated with 10 μg/mL of CHX for 6 h in the presence or absence of absence of BI 2536 (100nM). Endogenous REST, SCYL1, and β-actin levels were determined by western blot analysis. The images are representative of 2 independent experiments performed on 3 different independently derived clones for each genotype. (B) Quantification of REST protein levels in cell lines Hek293T and Hek293T-<i>SCYL1KO</i>. The ratio of the band intensity of REST to β-actin was normalized to the ratio of untreated (time = 0 h). Data represent mean ± standard error of the mean. (C) Flow cytometric analysis of cell cycle with propidium iodide DNA staining of exponentially growing Hek293T (n = 3) and Hek293T-SCYL1KO (n = 3) cells treated for 6 h with 100nM BI 2536 or DMSO. In the presence of BI 2536, cells accumulated to 4N DNA content, indicative of G2/M arrest. Data represent mean ± standard error of the mean. (D) Four distinct cultures of MDA-MB-231 cells were incubated with DMSO or BI2 2536 (100nM) for 6 h. Endogenous REST, SCYL1, and β-actin levels were determined by western blot analysis. (E) Quantification of REST protein levels in MDA-MB-231 cells. The ratio of the band intensity of REST to β-actin was normalized to the ratio of DMSO treated cells. Data represent mean ± standard error of the mean.</p

REST protein turnover and expression in Hek293T and Hek293T-<i>SCYL1</i>KO cells.

<p>(A) REST protein turnover in Hek293T cells. Hek293T-SCYL1KO cell lines (n = 3) were generated by using CRISPR-Cas9 technology (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178680#sec002" target="_blank">Materials and Methods</a>). Three Hek293T cell lines in which <i>SCYL1</i> was unaltered (Hek293T) and three Hek293T-SCYL1KO cell lines were seeded at a density of 250,000 cells per well of 6-well plates. The following day, the cells were treated with 10 μg/mL of CHX for the indicated time. Endogenous REST, SCYL1, and β-actin levels were determined by western blot analysis. The images are representative of 2 independent experiments performed on 3 different independently derived clones for each genotype. (B) Quantification of REST protein levels in cell lines Hek293T and Hek293T-<i>SCYL1KO</i>. The ratio of the band intensity of REST to β-actin was normalized to the ratio of untreated (time = 0 h). Data represent mean ± standard error of the mean.</p

REST turnover and expression in SCYL1-depleted Hek293T cells.

<p>Hek293T (A) or MDA-MB-231 (B) cells were transfected with or without RNAi duplexes targeting <i>SCYL1</i> (RNAi-1: SCYL1HSS126245, RNAi-2: SCYL1HSS183826, RNAi-3: SCYL1HSS183827). At 48 h post- transfection, cells were treated with 10 μg/mL of CHX for the indicated time. Endogenous REST, SCYL1, and β-actin levels were determined by western blot analysis. Comparable knockdown efficiencies were achieved for all 3 RNAi duplexes.</p

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. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice

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. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness