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.

WITHDRAWN: Effects of various glycerol concentrations and thawing temperatures on CASA parameters and acrosomal integrity of frozen–thawed canine spermatozoa

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause.The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy

The starburst cluster westerlund 1: The initial mass function and mass segregation

Westerlund 1 is the most important starburst cluster in the Galaxy due to its massive star content. We have performed BVIC and JKS photometry to investigate the initial mass function (IMF). By comparing the observed color with the spectral-type-intrinsic-color relation, we obtain the mean interstellar reddening of 〈E(B-V)〉 = 4.19 ± 0.23 and 〈E(J-KS )〉 = 1.70 ± 0.21. Due to the heavy extinction toward the cluster, the zero-age main sequence fitting method based on optical photometry proved to be inappropriate for the distance determination, while the near-infrared photometry gave a reliable distance to the cluster - 3.8 kpc from the empirical relation. Using the recent theoretical stellar evolution models with rotation, the age of the cluster is estimated to be 5.0 ± 1.0 Myr. We derived the IMF in the massive part and obtained a fairly shallow slope of Γ = -0.8 ± 0.1. The integration of the IMF gave a total mass for the cluster in excess of 5.0 × 104 M⊙. The IMF shows a clear radial variation indicating the presence of mass segregation. We also discuss the possible star formation history of Westerlund 1 from the presence of red supergiants and relatively low luminosity yellow hypergiants

UKIRT Widefield Infrared Survey for Fe+

The United Kingdom Infrared Telescope (UKIRT)Widefield Infrared Survey for Fe+ (UWIFE) is a 180 deg2 imaging survey of the first Galactic quadrant (7° &lt; l &lt; 62° |b| &lt;1°.5) that uses a narrow-band filter centred on the [Fe II] 1.644-μm emission line. The [Fe II] 1.644-μm emission is a good tracer of dense, shock-excited gas, and the survey will probe violent environments around stars: star-forming regions, evolved stars, and supernova remnants, among others. The UWIFE survey is designed to complement the existing UKIRTW idefield Infrared Survey for H2 (UWISH2). The survey will also complement existing broad-band surveys. The observed images have a nominal 5Ï? detection limit of 18.7 mag for point sources, with a median seeing of 0.83 arcsec. For extended sources, we estimate a surface brightness limit of 8.1 Ã? 10-20 W m-2 arcsec-2. In this paper, we present an overview and some preliminary results of this survey. © 2014 The Authors Published by Oxford University Press on behalf of the Royal Astronomical Society

Akabane viral encephalitis in calves in South Korea

This work was supported by the Brain Korea 21 Project and the Ministry of Agriculture and Forestry (399002-3), Republic of Korea

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