Transposons-based genetic strategies have been established in vertebrate species over the last decade. The major advantage of these strategies is that they facilitate analysis of gene function in an easy, controlled, and scalable manner, thereby avoiding some of the limitations of the classical DNA microinjection method. At Creative Biolabs, we use transposons as gene transfer vectors to generate transgenic models with high efficiency of chromosomal integration and single copy insertion.
Introduction of Transposons
Transposons are sequences of DNA that can move around in a genome. Generally, transposons are divided into two types: retrotransposons (class I) and DNA transposons (class II). The ‘copy-and-paste' retrotransposon is mobilized by transcribing an RNA copy, which is then reverse transcribes and integrated into the genome. However, the DNA transposon, a ‘cut-and-paste' model, is mediated by directly cutting DNA.
Introduction of Transposon-Mediated Gene Delivery
Three general uses of transposon vectors for the generation and manipulation of transgenic animals have been explored: germline transgenesis, somatic cell transgenesis/gene therapy, and random germline insertional mutagenesis. First of all, germline transgenesis by transposition is achieved by co-delivery of in vitro transcribed mRNA encoding the transposase and transposon vector DNA into the early embryos of the mouse. This method shows the advantages of generating offspring with multiple independent insertions but the disadvantages of very low expression in some cases. Prior to the construction of a vector, important considerations such as tolerance for cargo size of the transposon vector, integration site preference, and expression tendency need to be made to ensure successful delivery. In addition, transgenes can also be introduced into somatic cells of animals or randomly inserted to cause germline mutations, mainly for human gene therapy and genetic studies, respectively.
Fig.1 Class I and class II transposable elements.
(Ivics et al. 2009)
Multiple transposons now have been shown to be active in vertebrates, here we utilize three most commonly used transposable elements for the creation of transgenic mouse models:
Tol2 belongs to the Hat family and is a naturally occurring active ‘cut-and-paste' transposon. One advantage of Tol2 for germline mutagenesis is the capacity to transpose very large vectors. Besides, Tol2 transposon has little integration site preference and shows even higher transgenic efficiency compared to Sleeping Beauty and PiggyBac.
Sleeping Beauty belongs to the Tc1/mariner family, a type of ‘cut-and-paste' transposable elements. It is a synthetic transposon and the first vertebrate transposable element reconstructed from defective endogenous elements. Germline transgenesis using SB is achieved by co-injecting vector DNA with in vitro transcribed mRNA encoding the SB10 transposase into a one-cell mouse embryo pronuclei.
With a broad host range, the PiggyBac transposon is currently the most widely used transposon system for genetic manipulations. By now, this transposon has been well characterized and it represents a number of unique characteristics. Besides, one biggest advantage of PB is its precise excision.
Additionally, many other model creation technologies listed below are available from Creative Biolabs.
Supported by a group of seasoned experts who have mastered the most sophisticated technologies for animal model creation, Creative Biolabs is willing to work with you for a custom design to fit your exact requirements. Contact us or inquire us for more information or a formal quote if you are interested.
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