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

There has been considerable effort to uncover genetic and biochemical influences on bovine lactational traits using in vitro cell models of mammary tissue. However, modelling of biological processes in cultured cells, particularly those isolated from terminally differentiated tissues, is challenging. Primary cells can quickly lose their representative gene expression profiles and immortalized cells do not often retain their physiological relevance. In this article, we present a combined protocol, which describes the culture and characterisation of primary bovine mammary epithelial cells (pbMECs) and their transfection and editing, using the critical gene for lactogenesis, DGAT1, as an example of this methodology. We demonstrate how fluorescence-activated cell sorting (FACS) can greatly improve the frequency of edited cells in the final cell population. We also show that growth of the sorted CRISPR-Cas9-edited pbMCEs on Matrigel improves the maintenance of lactogenic gene expression. The combination of these methodologies opens the window for the testing of gene variant function in primary cell lines.

Janelle Moody, Emily Mears, Alexander J Trevarton, Marita Broadhurst, Adrian Molenaar, Thaize Chometon, Thomas Lopdell, Matthew Littlejohn, and Russell Snell. Successful editing and maintenance of lactogenic gene expression in primary bovine mammary epithelial cells. In Vitro Cellular & Developmental Biology-Animal, 59:316-330, 2023. https://link.springer.com/article/10.1007/s11626-023-00762-6

Left: Prof. Dennis Thomas T., Right: Aswathi NV., Department of Plant Science, Central University of Kerala, Tejaswini Hills, Periye (P.O.), Kasaragod, Kerala 671320, India

**Transverse thin cell layer (tTCL) technology: a promising tool for micropropagation of Centratherum punctatum Cass.**

Thin cell layers (TCLs) are very tiny explants originating from any plant tissue or organ with their size only reaching some millimeters. The TCLs can be excised either longitudinally (lTCL) or transversely (tTCL) from the source. Since the first report of the use of TCL explants in tobacco pedicel by Tran Thanh Van in 1973, this technique has gained serious attention among researchers. TCL culture is expedient in terms of overall plantlet production compared to traditional methods. The use of minimal explant tissue with extremely smaller size makes them highly sensitive to the culture conditions and the culture medium. TCL technique can be employed for the successful micropropagation of elite plants for their rapid propagation for increased supply to mankind. It also helps to propagate rare and endangered medicinal plants for an unlimited continuous supply of plants as well as important secondary metabolites. Centratherum punctatum is an important medicinal plant with a wide range of pharmacological properties like antimicrobial, antioxidant, anti-tumor, anti-inflammatory, anti-plasmodial, HIV reverse transcriptase, cytotoxic and wound healing activities. tTCL culture technique is applied to standardize efficient high-frequency micropropagation of C. punctatum in the present study. We observed that plant growth regulators (PGRs), the thickness of tTCLs, and the source of tTCLs have a significant effect on direct shoot regeneration. 1.0 mm and 2.0 was the best size 1.5 mg L−1 TDZ and 1.0 mg L−1 BAP + 0.2 mg L−1 NAA was the best PGRs for leaf and nodal tTCLs respectively. The percentage of response was observed to be as high as 96% and 87% for leaf and node tTCLs. Clonal fidelity assessment by SCoT markers and Chlorophyll content-based assessment of acclimatization success are also described. This will aid in the mass production of the plant for pharmaceutical usage and also paves a path towards the micropropagation of related members of its family Asteraceae.
NV Aswathi and TD Thomas. Transverse thin cell layer (tTCL) technology: a promising tool for micropropagation of Centratherum punctatum Cass. In Vitro Cellular & Developmental Biology-Plant, 59:340-353, 2023. https://doi.org/10.1007/s11627-023-10348-2