Primary transgenic bovine cells and their rejuvenated cloned equivalents show transgene-specific epigenetic differences.

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine ß-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences.

A virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibition.

Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus-free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling ('2i'). Non-invasive real-time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthesis and did not expand after passaging. Following transfection, non-proliferative primary colonies expressed discriminatory markers of pluripotency, including endogenous iPSC factors, CDH1, DPPA3, NANOG, SOCS3, ZFP42, telomerase activity, Tra-1-60/81 and SSEA-3/4, but not SSEA-1. This indicates that they had initiated a self-sustaining pluripotency programme. Bovine iPSC-like cells maintained a normal karyotype and differentiated into derivatives of all three germ layers in vitro and in teratomas. Our study demonstrates that conversion into induced pluripotency can occur in quiescent cells, following a previously undescribed route of direct cell reprogramming. This identifies a major species-specific barrier for generating iPSCs and provides a chemically defined screening platform for factors that induce proliferation and maintain pluripotency of embryo-derived pluripotent stem cells in livestock.

Gene targeting from laboratory to livestock: current status and emerging concepts.

The development of methods for cell-mediated transgenesis, based on somatic cell nuclear transfer, provides a tremendous opportunity to shape the genetic make-up of livestock animals in a much more directed approach than traditional animal breeding and selection schemes. Progress in the site-directed modulation of livestock genomes is currently limited by the low efficiencies of gene targeting imposed by the low frequency of homologous recombination and limited proliferative capacity of primary somatic cells that are used to produce transgenic animals. Here we review the current state of the art in the field, discuss the crucial aspects of the methodology and provide an overview of emerging approaches to increase the efficiency of gene targeting in somatic cells.

Direct determination of the influence of extreme temperature on transposition and structural mutation rates of Drosophila melanogaster mobile elements.

Two sets of mutation accumulation lines, one reared at 28 degrees C and the other at 24 degrees C, were compared for their transposition and rearrangement rates of eleven transposable element families. The changes affecting mobile elements were analysed by the Southern technique and in situ hybridization. No differences were found between treated and control lines. The role of the host genotype in transposition control and the significance of structural mutations in transposable element dynamics are discussed.

Structural heterogeneity and genomic distribution of Drosophila melanogaster LTR-retrotransposons.

Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, beta-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.