Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos.

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome.

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk.

Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG.

DNA oligonucleotides and plasmids perform equally as donors for targeted gene conversion.

Site-specific gene modifications in cells are initiated by the introduction of exogenous DNA. We used a recently established cell assay to compare the ability of DNA donors to induce a single point mutation that converts a target gene encoding blue fluorescent protein (BFP) into expressing green fluorescent protein (GFP). In a chromosomal assay with cells stably expressing BFP, we showed that fluorescently labeled single-stranded oligonucleotides and a donor plasmid cotranscribing a red fluorescent protein provide similar efficiencies in triggering BFP-GFP conversions.