PolyA Trapping & Non-Sense Mediated Decay
The most commonly used gene trap strategy is promoter gene trapping, which involves a gene trap vector containing a promoterless selectable marker cassette such as bgeo (shown below). In promoter gene trapping, the mRNA of the selectable marker gene can be transcribed only when the gene trap vector inserts within a transcriptionally active gene. Although promoter trapping is effective at inactivating genes, inserts within transcriptionally silent loci cannot be selected by this strategy.
It is believed that 40-50% of the genome is transcriptionally inactive in ES cells, suggesting that this component of the genome is inaccessible to promoter gene trap vectors. To capture a broader spectrum of genes including those not expressed in ES cells, polyA trap vectors have been developed in which a constitutive promoter drives the expression of a selectable marker gene lacking a polyA signal. Replacing the polyA signal is a splice donor site designed to splice into downstream exons. In this strategy, the mRNA of the selectable marker gene can be stabilized upon trapping of a polyA signal of an endogenous gene regardless of its expression status in the target cells.
We have found that polyA trap vectors do indeed trap a higher proportion of unique genes compared to general promoter gene trap vectors. We have discovered that polyA gene trap insertions, often occur in the 3’ end of genes. This occurs because most insertions in the 5’ exons activate nonsense mediated decay (NMD) of the antibiotic resistance fusion transcript, leading to an inability to select for 5’ insertions. NMD is an mRNA-surveillance mechanism universally conserved among eukaryotes, which is responsible for the degradation of mRNAs with potentially harmful nonsense mutations. It is activated by termination codons more than 55 bp upstream of the final splice junction site.
An insertion within the 3’ end of a gene will often have limited mutagenic potential. As an interim solution, the CMHD generated a complement of gene trap vectors in which the polyA site of beta galactosidase reporter was deleted. Gene trap insertions with these vectors (see PolyA Trap Vectors page) results in the generation of unstable trapped transcripts, leading to hypomorphic mutations but are unlikely to generate null mutations.
Although insertions within the final intron of genes are disadvantages from a mutagenesis standpoint, it does offer improved functionality as an expression marker. The 3’-insertions of gene trap vectors containing a fluorescent reporter allows high-throughput random protein tagging which can be used to resolve expression profiles at the subcellular level – an important and useful tool for the characterization of new genes. We wanted to maintain this functionality in our gene trap vector design while still allowing mutagenesis of the trapped gene. To achieve this we are developing a novel set of “transcript trapping” vectors that utilize nonsense-mediated decay of nascent transcripts as a mutagenesis agent.
The NAIST group overcame the challenge of NMD by developing a novel vector known as UPATrap (shown below) that effectively suppresses NMD by introducing a floxed internal ribosome entry site (IRES) sequence upstream of initiation codons in all three reading frames inserted between the NEO gene and the splice donor sequence of the conventional RET polyA trap vector (Ishida 2005 reference). The resulting bicistronic message escapes NMD and is translated. To increase mutagenicity Cre recombination can be performed after selection and cloning in vitro or in vivo to prevent expression of the 3’ portion of the trapped gene.
The IRES sequence inserted downstream of the termination codon of the NEO gene prevented activation of NMD, allowing trapping of transcriptionally silent genes without a bias in the vector-integration site. Thus, our facility utilizes two complementary approaches to overcome the limitations of NMD, thereby developing mutagenic polyA trap gene trap vectors.