You transform a population of cells with a transgene and isolate a cell line that has integrated the gene into its genome, but in which the gene is not expressed. Speculate as to what may be preventing the gene from being expressed. Describe two ways to test this possibility.

The transgene could be silenced by: 1- epigenetic mechanisms (e.g., DNA methylation, histone deacetylation, etc.), and 2-the RNA interference (RNAi) pathway

Explanation:

Epigenetics refers to the heritable changes caused by the activation and deactivation of genes without changing the DNA sequence. Epigenetic mechanisms include 1-DNA methylation on cytosine residues, and 2-modifications on amino acids in the tails of histone proteins (e.g., methylation, acetylation, ribosylation, phosphorylation, etc). These two epigenetic mechanisms can modify DNA structure and thus alter the accessibility of transcription factors, thereby suppressing gene/transgene expression. Moreover, the RNA interference pathway (RNAi) is a naturally occurring mechanism capable of suppressing gene/transgene expression both at transcriptional and post-transcriptional levels. The RNAi mechanism is triggered by regulatory non-coding RNAs (ncRNAs) such as, among others, long non-coding RNAs (lncRNAs) and small ncRNAs (e.g., miRNAs, piRNAs, siRNAs, etc). In this case, for example, DNA methylation on promoter sequences could suppress transgene expression, thereby methods to measure DNA methylation can be used to test this possibility (e.g., bisulfite genomic sequencing). Moreover, miRNAs that bind to the messenger RNA of the transgene could also inhibit its expression by triggering the RNAi pathway (e.g., mRNA degradation), thereby methods to measure mRNA levels can be used to test this possibility (e.g., RT-PCR).