Deciphering the role of MFGE8 in lactation biology using CRISPR- CAS9 based gene editing in Buffalo mammary epithelial cells

Abstract

Abstract Milk fat globule EGF factor 8 (MFGE8) also known as Lactadherin is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group has previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we have generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation biology. Among three gRNAs used to generate knock-outs, gRNA3 reduced MFGE8 expression with better efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. To comprehend the role of MFGE8, mfge8-/-/gRNA3 cells were examined for proteome level changes in comparison to wild type cells using an iTRAQ experiment. A total 4282 proteins were identified in mfge8-/-/gRNA3 cells and among them 178 were found to be differentially expressed above and below a threshold of ≥1.5 and ≤0.6. Majority of DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores the downstream effects of disruption of MFGE8 gene. Overall, the present study will provide new insights into lactation biology.