Deciphering Mammalian Biology and Disease

Large-Scale Transposon Insertional Mutagenesis in Mice

One of the most direct applications of the PB system is for insertional mutagenesis. We have now developed an efficient PBtransposition system in mice, which can be used to rapidly produce large numbers of single PB insertional mutant strains. This technical achievement allows the quick and effortless correlation between an insertion, the gene disrupted, and the phenotype. Insertional mutagenesis is triggered by the simple breeding of a PBase-expressing transgenic line with a line carrying a single non-autonomous PB transposon, which eliminates the costly and labor-intensive production of mutants by ES cell-based knockout technology.

Furthermore, the genotypes of the offspring can be quickly visually identified as endowed by coat color-labeled PBase transgene and RFP labeledPB transposon (PB[RFP]). The use of RFP to mark the transposon achieves two additional design goals: 1) PB insertions mobilized into new locations will exhibit a color different from the original insertion, and 2) homozygous mutants can be visibly distinguished from heterozygous or wild-type siblings. 

Visual determination of the genotypes of the mice greatly simplifies phenotypic characterization, colony management, breeding, and dramatically reduces the animal work cost. The disrupted genes can be simply identified by PCR. The transposon could also carry a LacZ reporter. The loxP sites carried by these insertions can also be used to generate deletions and chromosome rearrangements. Finally, PB insertions can be precisely excised from their original sites in the genome, allowing the reversion of insertional mutations. 

We have produced more than 500 PB insertions and found that they are widely distributed throughout the genome. Such non-biased transposition events are essential for large scale mutagenesis. Approximately 50% of the insertions land in transcription units. Characterization of the first 100 insertions within transcription units reveals that about 20% cause embryonic lethality or other phenotypes. 

We examined insertions in five previously knocked out genes and found that four of them exhibit similar phenotypes while the other one produces a phenotype weaker than its corresponding knockout. These results demonstrate the PBtransposon to be a highly efficient mutagen. In addition, some insertions in genomic regions with no predicted genes also display phenotypes, suggesting that PB could also be a powerful tool for interrogating important genetic elements such as regulatory sequences and microRNA genes. The PB transposon system, thus, permits for the first time the efficient production of a genome-wide set of insertional mouse mutants, providing a unique opportunity to use forward genetics to decipher mammalian biology and disease.