Successful editing and maintenance of lactogenic gene expression in primary bovine mammary epithelial cells.

In vitro investigation of bovine lactation processes is limited by a lack of physiologically representative cell models. This deficiency is most evident through the minimal or absent expression of lactation-specific genes in cultured bovine mammary tissues. Primary bovine mammary epithelial cells (pbMECs) extracted from lactating mammary tissue and grown in culture initially express milk protein transcripts at relatively representative levels. However, expression drops dramatically after only three or four passages, which greatly reduces the utility of primary cells to model and further examine lactogenesis. To investigate the effects of alternate alleles in pbMECs including effects on transcription, we have developed methods to deliver CRISPR-Cas9 gene editing reagents to primary mammary cells, resulting in very high editing efficiencies. We have also found that culturing the cells on an imitation basement membrane composed of Matrigel, results in the restoration of a more representative lactogenic gene expression profile and the cells forming three-dimensional structures in vitro. Here, we present data from four pbMEC lines recovered from pregnant cows and detail the expression profile of five key milk synthesis genes in these MECs grown on Matrigel. Additionally, we describe an optimised method for preferentially selecting CRISPR-Cas9-edited cells conferring a knock-out of DGAT1, using fluorescence-activated cell sorting (FACS). The combination of these techniques facilitates the use of pbMECs as a model to investigate the effects of gene introgressions and genetic variation in lactating mammary tissue.

Correction: Surveying the spatial distribution of feral sorghum (Sorghum bicolor L.) and its sympatry with johnsongrass (S. halepense) in South Texas.

[This corrects the article DOI: 10.1371/journal.pone.0195511.].

Surveying the spatial distribution of feral sorghum (Sorghum bicolor L.) and its sympatry with johnsongrass (S. halepense) in South Texas.

Sorghum (Sorghum bicolor) is an important grain and forage crop grown across the US. In some areas, sorghum can become feral along roadsides and other ruderal areas, as a result of seed spill during harvest or transport. In some of these situations, feral sorghum grows in or near established johnsongrass (S. halepense) populations. Johnsongrass, a wild relative of sorghum and an incredibly noxious weed, is capable of hybridizing with cultivated sorghum. Because commercial hybrid sorghum cultivars are produced with cytoplasmic male sterility, progeny of the hybrid crop which compose the founder feral populations also segregate for male sterility. Consequently, male sterility in feral sorghum may increase the risk of outcrossing with johnsongrass. Using field surveys and spatial modelling, the present study aimed at documenting the occurrence of feral sorghum and understanding the anthropogenic and environmental factors that influence its distribution. Further, this research documented the sympatry of feral sorghum and johnsongrass in the roadside habitat. A total of 2077 sites were visited during a systematic field survey conducted in fall 2014 in South Texas. Feral sorghum and johnsongrass were found in 360 and 939 sites, while the species co-existed at 48 sites (2.3% of all surveyed sites). The binary logistic analysis showed a significant association between the presence of feral sorghum and road type, road body-type, micro-topography of the sampling site, nearby land use, and the presence of johnsongrass, but no association with the distance to the nearest grain sorting facility. The probability of finding feral sorghum away from johnsongrass patches was generally higher than finding them co-occur in the same location. A probability map for spatial distribution of feral sorghum was developed using the nearby land use type and the regional habitat suitability for johnsongrass as two key predictors. Overall, results show that feral sorghum and johnsongrass co-occur at low frequencies in the roadside habitats of South Texas, but these low levels still present a significant opportunity for hybridization between the two species outside of cultivated fields.

Bronchodilator response in relation to beta2-adrenoceptor haplotype in patients with asthma.

RATIONALE: Genetic variation of the beta2-adrenoceptor (ADRB2) influences receptor function in vitro. There are reports that, in vivo, bronchodilator response is related to ADRB2 genotype, and that clinical outcomes during chronic therapy with beta2-agonist drugs are also influenced by genotype. Whether these features are related to single nucleotide polymorphisms or to combinations (haplotypes) is unclear. OBJECTIVES: Our aim was to measure bronchodilator response in patients with asthma stratified by ADRB2 haplotype. This was done after eliminating the confounding effect of prior drug treatment with inhaled beta2-agonists and corticosteroids. METHODS: ADRB2 haplotype was determined in 176 patients with asthma, of whom 161 harbored the six most common combinations. Treatment with inhaled beta2-agonists and inhaled corticosteroids was withheld for appropriate intervals. Spirometric changes 20 minutes after a single dose of albuterol (2.5 mg by nebulizer) were then recorded. RESULTS: There were no significant differences in bronchodilator response (% improvement in FEV(1)) with respect to any of the major ADRB2 haplotypes or genotypes. CONCLUSIONS: Genetic variation of the ADRB2 does not influence the immediate response to inhaled beta2-agonist. The confounding effect of tolerance resulting from regular beta2-agonist use must be controlled when assessing the pharmacogenetic influences on clinical outcomes with beta2-agonists.