From left to right: Leonie Walter (first and corresponding author), Richard Fry, Amy Logan and Brian Leury.

Investigation on the suitability of milk-derived primary bovine mammary epithelial cells grown on permeable membrane supports as an in vitro model for lactation

Primary cell culture is a powerful tool to investigate fundamental processes behind milk production in the mammary gland. The traditional approach to obtain cells for in vitro experiments is to collect mammary tissue from an abattoir or from mammary biopsies. Using mammary tissue harvested from abattoirs has the drawback of limiting the targeted selection of individual animals. Likewise, the limiting factor for mammary biopsies are ethical considerations associated with this invasive procedure. An alternative method is to harvest mammary epithelial cells (MECs) from the milk of lactating animals. This technique has the added advantage of providing cultures of MECs without contamination from other cell types, particularly fibroblasts. In this study we investigated the functionality of milk-derived primary bovine mammary epithelial cells (pbMECs) as an in vitro model for lactation. Functionality of these cells was assessed in terms of intracellular triacylglycerol content and the gene expression of kappa casein (CSN3), a milk protein, and diacylglycerol acyltransferase (DGAT1), a key enzyme in triacylglycerol synthesis. We compared the functionality of milk-derived pbMECs to cultures obtained from mammary tissue. Moreover, we tested a 3D-like cell culture method, where pbMECs are grown on polyester membrane (Transwell) inserts suspended in a cell culture vessel, thus creating an environment similar to the in vivo environment, with an apical and basal compartment above and below the membrane insert. We hypothesized that this technique could be particularly useful for pure cultures of pbMECs derived from milk. In order to assess the suitability of this technique for milk-derived pbMECs we compared the Transwell culture to 3D culture on an extracellular matrix gel and 2D culture on a plastic surface. Overall, 3D culture remained the most suitable environment in terms of CSN3 and DGAT1 gene expression and intracellular triacylglycerol production. However, the results of our study partially confirm our hypothesis that Transwell culture is particularly suitable for milk-derived MEC, with the highest viability of milk derived cells under these conditions and increased DGAT1 expression compared to the 2D culture environment. We suggest that this technique, with further optimization, could be suitable for the in vitro study of lactation physiology.

Leonie Walter, Richard Fry, Amy Logan, Brian J. Leury.  Investigation on the suitability of milk-derived primary bovine mammary epithelial cells grown on permeable membrane supports as an in vitro model for lactation.  In Vitro Cellular & developmental Biology-Animal 56: 386 – 398, 2020.

Left to right: Atsushi Kunii, Tetsushi Sakuma, Takashi Yamamoto

Various strategies of effector accumulation to improve the efficiency of genome editing and derivative methodologies

Various genome editing systems have been established and sophisticated in several decades with two concepts: chimeric protein of a DNA-binding domain and a nuclease domain, and RNA-guided CRISPR-Cas nuclease. Chimeric nucleases such as zinc finger nuclease (ZFN) and transcriptional activation-like effector nuclease (TALEN) compose of the single pairs of the proteins; therefore, the expandability of their functionality is somewhat limited. To implement some additional functions in these proteins, the corresponding effector domains should be directly fused or recruited via polypeptide tags. On the other hand, CRISPR-Cas platforms contain both protein and RNA within their functional complex. It means that CRISPR-Cas has a superior property in terms of its extended performance. In addition to the direct fusion and polypeptide tag-mediated recruitment to Cas9 protein, sgRNA enables various RNA aptamer-assisted accumulation of effector molecules. Moreover, the combination and/or hierarchical organization of these multiple types of accumulable systems can further increase the capacity to pile up the effectors. In our review article, we summarized the current situation and future perspectives of CRISPR-based effector accumulation strategies and a wide variety of their applications. Such accumulative systems have been utilized not only for genome editing purposes but also for the derivative approaches including chromatin visualization, transcriptional control, and epigenome editing. Over the simple genetic engineering such as gene knockout and knock-in, the advanced CRISPR systems reviewed herein will bring an era of “Genome Editing 2.0,” enabling efficient, scalable, and variable editing, writing, isolating, detecting, and labeling of DNA, RNA, and their modifications.

Atsushi Kunii, Takashi Yamamoto, Tetsushi Sakuma. Various strategies of effector accumulation to improve the efficiency of genome editing and derivative methodologies.  In Vitro Cellular & Developmental Biology-Animal 56:359-366, 2020.