In silicon analysis of transcriptome mutation from immortalized porcine alveolar macrophage

Abstract

The immortalization of a cell is a process in which a cell is transformed from its primary state to the immortal one. Likewise, numerous immortalized cell lines of many living organisms have been produced to maintain the cell’s characteristics for long-term projects. Additionally, understanding the genetic mechanism related to immortalization can provide further insights to improve the quality of immortal cells. However, the genetic basis of immortalizing process in pigs has not been completely clarified yet as well as its transcriptome profiles. Nevertheless, RNA sequencing has made it possible to widen our horizons of the transcriptome by studying gene expression, confirming pathogenicity and detecting transcript alterations. Hence, in this project, we performed the variant identification of two primary alveolar macrophages (PAM) compared with two immortalized porcine alveolar macrophages (iPAM) which were transduced with two exogenous genes (SV40LT and hTERT) and predicted its consequences involving immortalizing process. RNA sequencing was used for single nucleotide polymorphisms (SNPs) and short insertions and deletions (INDELs) identifying in 2 porcine cell lines (immortal; iPAM61 and iPAM303 versus mortal; PAM61 and PAM303). Using an in-house pipeline, a total of 20,194 known RNA-seq variants were genotyped, of which a variety of variants were exclusively found in iPAMs. In addition, functional analysis was carried out using DAVID bioinformatic tool. Interesting missense gene variants and mutations located in coding regions were explored in many genes related to mismatch repair such as: LIG1; MSH2; RFC2; PMS2. All in all, we did not identify any gene mutations in coding regions related to the development of immortalization, we predicted that non-coding areas might have effect on that process.