|Handler, Alfred - Al|
|Fraser, Jr., Malcolm|
Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2004
Publication Date: 1/1/2005
Citation: Li, X., Harrell, R.A., Handler, A.M., Beam, T., Hennessy, K., Fraser, Jr., M.J. 2005. Terminal region proximal internal domain sequences of the piggyBac transposon are necessary for efficient transformation of target genomes. Insect Molecular Biology. 14:17-30. Interpretive Summary: The ability to achieve gene transfer in economically important insects is a major goal of our laboratory at the CMAVE. Development of this methodology depends upon the discovery and analysis of efficient and stable gene transfer vector systems. Previously, the piggyBac vector from Trichoplusia ni was found to mediate germline transformation in several species spanning four orders of insects. In order to improve and expand the use of the piggyBac system, as well as to better understand its mechanism of transposition, a systematic study was used to determine the minimal terminal DNA sequences required for mobility. It was discovered that 172 base pairs from the right 3'-terminal sequence and 276 base pairs from the left 5'-terminal sequence were sufficient for efficient vector transpostion. Since shorter transposon vectors transform more efficiently than longer vectors, this information will be useful to the creation of new highly efficient insect vector systems.
Technical Abstract: A previous report demonstrated that deletion mutations of the entire piggyBac internal domain had no effect on interplasmid transposition in microinjected embryos. In applying internal domain deleted vectors to transformations of insects we noted a significant decline in the efficiency of transformation compared to full length or less extensive internal domain deletion constructs. We have reexamined the deletion mutation analysis of internal domain sequences using a PCR strategy to construct selective alterations that effectively add terminal repeat proximal internal domain (ID) sequences to each terminus. A new series of piggyBac ID deletion plasmids containing the 3xP3-ECFP marker gene were compared for germ-line transformation of Drosophila melanogaster. Based on these results we constructed a new minimal piggyBac transposon cartridge (ITR1.1k) having 276 bp of the 3' or 172 bp 5' ID sequences in addition to the 3' and 5' terminal repeat regions. This cartridge was demonstrated to retain wild- type levels of insect transformation efficiency in two separate experimental trials. Southern hybridization analysis confirmed the presence of the piggyBac transposon sequences, while insertion site analyses confirmed the integrations targeted TTAA sites. These analyses identify the ID sequences proximal to each terminal repeat configuration that are important in movement of the vectored sequences from plasmids into the insect genome, even though they are not required for excision or interplasmid transposition. We also constructed a traditional minimal piggyBac transposon vector pXL-BacII-ECFP containing these ID sequences that yields a high transformation frequency. During our analysis we discovered a point mutation in the internal repeat domain of the piggyBac 3' terminal repeat that appears to be the result of a mis-incorporation during PCR amplification, but has no apparent effect on transformation efficiency.