Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

Research articl

Assessment of Artificial MiRNA Architectures for Higher Knockdown Efficiencies without the Undesired Effects in Mice.

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both -MG amiRNA cells and amiRNA low-expression cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Ohtsuka, M., Miura, H., Mochida, K. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015). https://doi.org/10.1186/s12864-015-1432-

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies

Dissecting Oct3/4-Regulated Gene Networks in Embryonic Stem Cells by Expression Profiling

POU transcription factor Pou5f1 (Oct3/4) is required to maintain ES cells in an undifferentiated state. Here we show that global expression profiling of Oct3/4-manipulated ES cells delineates the downstream target genes of Oct3/4. Combined with data from genome-wide chromatin-immunoprecipitation (ChIP) assays, this analysis identifies not only primary downstream targets of Oct3/4, but also secondary or tertiary targets. Furthermore, the analysis also reveals that downstream target genes are regulated either positively or negatively by Oct3/4. Identification of a group of genes that show both activation and repression depending on Oct3/4 expression levels provides a possible mechanism for the requirement of appropriate Oct3/4 expression to maintain undifferentiated ES cells. As a proof-of-principle study, one of the downstream genes, Tcl1, has been analyzed in detail. We show that Oct3/4 binds to the promoter region of Tcl1 and activates its transcription. We also show that Tcl1 is involved in the regulation of proliferation, but not differentiation, in ES cells. These findings suggest that the global expression profiling of gene-manipulated ES cells can help to delineate the structure and dynamics of gene regulatory networks

Ellipsometric Study of Anodic Oxide Films on Titanium in Hydrochloric Acid, Sulfuric Acid, and Phosphate Solution

The anodic oxide film on titanium has been studied by ellipsometry and SEM observation. Ex situ multiple‐angle‐of‐incidence and in situ ellipsometric measurements allow the complex refractive index to be estimated at n = 2.3- 2.9i for the titanium substrate and at n = 2.1-0.03i for the anodic oxide film at wavelength 546.1 nm. The anodic oxide film thickness increases linearly with potential in a range from −0.55 to 7.5V (RHE) at the rate of 2.8 nm V^−1 in phosphate solutions of pH 1.6–12.1, 2.5 nm V^−1 in 0.1M HCl solution, and 2.4 nm V^−1 in 0.1M H2SO4 solution. At potentials more positive than 7.5V, the film breaks down, leading to the formation of a thick oxide film probably due to an increased ionic current through the breakdown sites. The film composition is estimated to be TiO2(H20)l.4 or TiO0.6(OH)2.8 , which suggests the presence of hydroxyl bridge in its bonding structure

Cathodic Reduction of Anodic Oxide Films Formed on Titanium

Cathodic reduction behavior of the anodic oxide film on titanium has been investigated by using ellipsometry combined with electrochemistry. In acidic sulfate solution, the anodic oxide film reductively dissolves into the solution as Ti(III) ion, resulting in the thinning of its thickness without any significant change of the optical property of the remaining film. In neutral phosphate solutiofi, the anodic oxide film absorbs hydrogen in the hydrogen evolution potential region, resulting in a change of the optical property without thinning its thickness. The amount of hydrogen absorbed per unit volume of the film does not depend on the film thickness but on the cathodic potential. The composition change estimated from measurements of anodJc charge during the hydrogen release process indicates that the hydrogen absorption begins to occur at about -0.25V (vs. RHE) and that the anodic film changes in its composition from TiO2 to TiOOH at -0.9V. The hydrogen absorption induces a decrease of the refractive index and an increase of the extinction index of the anodic film