DsRed gene expression by doxycycline in porcine fibroblasts and cloned embryos using transposon

To develop a transgenic pig, introduction of foreign genes into fibroblasts is required. In this study, Piggybac transposition was used to produce tetracycline dependent gene expressing cloned embryos. Red fluorescence proteins (DsRed) combined with tetracycline promoter flanked transposon sequences were transfected into fetal fibroblasts, and the transfected cells were used as the donor for somatic cell nuclear transfer. Induction of DsRed expression was successfully controlled by doxycycline treatment in donor fibroblasts and early stage embryos. In conclusion, this study suggested that Piggybac transposition could deliver genes into cells or embryos for developing transgenic pig.Keywords: Miniature pigs, transfection, Piggybac, somatic cell nuclear transfer (SCNT), RFPAfrican Journal of Biotechnology Vol. 12(21), pp. 3188-319

Improvement Characteristics of Ground Using C.G.S Through Field Case Study

Compaction Grouting System is widely used in densifying loose soils or fill voids caused by sinkholes, poorly compacted fills, and soft ground improvement. Also, it is used in preventing liquefaction, re-leveling settled structures, and using compaction bulbs as structural elements of minipiles or underpinning. But the effects of ground improvement depending on the type of soil must be studied in order to adopt in various soils. In this study, characteristics analysis of the ground improvement and the effectiveness of reinforcement were grasped by this study which shows applied ground by Compaction Grouting System in domestic 6 sites. After Compaction Grouting, strength characteristics of the ground are much better than before Compaction Grouting through the results of the standard penetration test, the dynamic cone penetration test, the vane test and laboratory test using performance Evaluation of Linear Regression. Especially improvement of strength was shown over 17% by Compaction Grouting through prediction formulas in sand

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (similar to 24 hours) or their diameter (< 20 mu m) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Integrated Piezoelectric AlN Thin Film with SU-8/PDMS Supporting Layer for Flexible Sensor Array

This research focuses on the development of a flexible tactile sensor array consisting of aluminum nitride (AlN) based on micro-electro-mechanical system (MEMS) technology. A total of 2304 tactile sensors were integrated into a small area of 2.5 × 2.5 cm2. Five hundred nm thick AlN film with strong c-axis texture was sputtered on Cr/Au/Cr (50/50/5 nm) layers as the sacrificial layer coated on a Si wafer. To achieve device flexibility, polydimethylsiloxane (PDMS) polymer and SU-8 photoresist layer were used as the supporting layers after etching away a release layer. Twenty-five mM (3-mercaptopropyl) trimethoxysilane (MPTMS) improves the adhesion between metal and polymers due to formation of a self-assembled monolayer (SAM) on the surface of the top electrode. The flexible tactile sensor has 8 × 8 channels and each channel has 36 sensor elements with nine SU-8 bump blocks. The tactile sensor array was demonstrated to be flexible by bending 90 degrees. The tactile sensor array was demonstrated to show clear spatial resolution through detecting the distinct electrical response of each channel under local mechanical stimulus. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.1

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

<p>Abstract</p> <p>Background</p> <p>Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in <it>E. coli </it>have been unsuccessful.</p> <p>Results</p> <p>Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in <it>E. coli </it>and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of <it>E. coli </it>culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (<it>k</it>_cat/<it>K</it>_<it>M</it>) of the precursor proteins by two orders of magnitude.</p> <p>Conclusion</p> <p>A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in <it>E. coli</it>. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.</p

Application of transposon systems in the transgenesis of bovine somatic and germ cells

Several DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS. In bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%). These results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type