Transposon mutagenesis
Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis.
[3] In the early 1940s, McClintock was studying the progeny of self-pollinated maize plants which resulted from crosses having a broken chromosome 9.
In the case of bacteria, transposition mutagenesis is usually accomplished by way of a plasmid from which a transposon is extracted and inserted into the host chromosome.
[5] Early transposon mutagenesis experiments relied on bacteriophages and conjugative bacterial plasmids for the insertion of sequences.
A newer technique called shuttle mutagenesis uses specific cloned genes from the host species to incorporate genetic elements.
The Sleeping Beauty transposon system (SBTS) is the first successful non-viral vector for incorporation of a gene cassette into a vertebrate genome.
[7] The mechanism of the SBTS is similar to the Tn5 transposon system, however the enzyme and gene sequences are eukaryotic in nature as opposed to prokaryotic.
Once the enzyme is bound to both ends of the transposon, the IR/DR sequences are brought together and held by the transposase in a Synaptic Complex Formation (SCF).
Although transposase is specific for TA dinucleotides, the high frequency of these pairs in the genome indicates that the transposon undergoes fairly random integration.
[8] As a result of the capacity of transposon mutagenesis to incorporate genes into most areas of target chromosomes, there are a number of functions associated with the process.
Colonies that underwent random transposition events were identified by BamHI digestion and Southern blotting using an internal IS1096 DNA probe.
Mutations leading to an attenuated phenotype were mapped by amplification of adjacent regions to the IS1096 sequences and compared with the published M. tuberculosis genome.
The PiggyBac (PB) transposon from the cabbage looper moth Trichoplusia ni was engineered to be highly active in mammalian cells, and is capable of genome-wide mutagenesis.
Transposons contained both PB and Sleeping Beauty inverted repeats, in order to be recognized by both transposases and increase the frequency of transposition.
Since the mouse is a model organism for the study of human physiology and disease, this research will help lead to an increased understanding of cancer-causing genes and potential therapeutic targets.
