CRISPR/Cas9 Editing of 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 investigate 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 knockout 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.

牛泌乳过程的体外研究受限于缺乏具有生理代表性的细胞模型。这一缺陷在体外培养的牛乳腺组织中泌乳特异性基因表达量极低甚至完全缺失的现象中体现得最为明显。从泌乳乳腺组织中分离并体外培养的原代牛乳腺上皮细胞（primary bovine mammary epithelial cells，pbMECs），初始阶段的乳蛋白转录本表达水平相对具有生理代表性。然而，仅传代3至4次后，其表达量便会急剧下降，极大地限制了原代细胞作为模型进一步探究泌乳发生过程的实用性。为探究pbMECs中的等位基因变体（包括对转录过程的影响），我们开发了将CRISPR-Cas9基因编辑试剂递送至原代乳腺细胞的方法，该方法可实现极高的编辑效率。我们还发现，将细胞培养在由基质胶（Matrigel）构成的模拟基底膜上时，可恢复更具生理代表性的泌乳相关基因表达谱，且细胞能在体外形成三维结构。本研究公开了从妊娠奶牛中分离得到的4株pbMEC系的数据，并详细描述了这些在基质胶上培养的细胞中5个关键乳合成基因的表达谱。此外，我们还描述了一种优化后的方法，可利用荧光激活细胞分选术（fluorescence-activated cell sorting，FACS）优先筛选出二酰甘油酰基转移酶1（DGAT1）基因敲除的CRISPR-Cas9编辑细胞。这些技术的结合使得pbMECs作为模型，用于探究泌乳乳腺组织中基因渐渗与遗传变异的影响成为可能。