Characterization of L1 retrotransposition with high-throughput dual-luciferase assays.

Recent studies employing genome-wide approaches have provided an unprecedented view of the scope of L1 activities on structural variations in the human genome, and further reinforced the role of L1s as one of the major driving forces behind human genome evolution. The rapid identification of novel L1 elements by these high-throughput approaches demands improved L1 functional assays. However, the existing assays use antibiotic selection markers or fluorescent proteins as reporters; neither is amenable to miniaturization. To increase assay sensitivity and throughput, we have developed a third generation assay by using dual-luciferase reporters, in which firefly luciferase is used as the retrotransposition indicator and Renilla luciferase is encoded on the same or separate plasmid for normalization. This novel assay is highly sensitive and has a broad dynamic range. Quantitative data with high signal-to-noise ratios can be obtained from 24- up to 96-well plates in 2-4 days after transfection. Using the dual-luciferase assays, we have characterized profiles of retrotransposition by various human and mouse L1 elements, and detailed the kinetics of L1 retrotransposition in cultured cells. Its high-throughput and short assay timeframe make it well suited for routine tests as well as large-scale screening efforts.

Plug and play modular strategies for synthetic retrotransposons.

Recent progress in L1 biology highlights its role as a major driving force in the evolution of mammalian genome structure and function. This coincides with direct confirmation of the preponderance of long interspersed elements in mammalian genomes at the nucleotide level by large scale sequencing efforts. Two assay systems have been prominently featured in L1 studies over the past decade, which are used to assess L1 activities in cultured cells and transgenic mice respectively. However, constructing retrotransposon assay vectors and subsequent mapping of integration sites remain technically challenging aspects of the field. Synthetic biology approaches have changed the playing field with regard to the strategic design of retrotransposons. To streamline the construction and optimization of synthetic retrotransposons, we have implemented a highly efficient modular design for L1 vectors allowing "plug and play" swapping of individual modules as new knowledge is gained and optimization of constructs proceeds. Seven functional modules are divided by strategically placed unique restriction sites. These are utilized to facilitate module exchange and construction of L1 vectors for gene targeting, transgenesis and cell culture assays. A "double SfiI" strategy utilizing two non-complementary overhangs allows insert swapping to be carried out with a single, robust restriction/ligation cycle. The double-SfiI strategy is generic and can be applied to many other problems in synthetic biology or genetic engineering. To facilitate genomic mapping of L1 insertions, we have developed an optimized inverse PCR protocol using 4-base cutters and step-down cycling conditions. Using this protocol, de novo L1 insertions can be efficiently recovered after a single round of PCR. The proposed modular design also incorporates features allowing streamlined insertion mapping without repeated optimization. Furthermore, we have presented evidence that efficient L1 retrotransposition is not dependent on pCEP4 conferred autonomous replication capabilities when a shortened puromycin selection protocol is used, providing a great opportunity for further optimization of L1 cell culture assay vectors by using alternative vector backbones.

Estrogen mediated inhibition of dopamine transport in the striatum: regulation by G alpha i/o.

In the current study, the interaction between estrogen priming and dopamine D2 receptor activation on dopamine uptake in the striatum of ovariectomized female rats was investigated. Basal ADP-[(32)P(i)]ribosylation of G(i/o) was examined in synaptosomal membranes prepared from ovariectomized, estrogen primed or N-p-(isothiocyanatophenethyl) spiperone (NIPS) treated rats. [(32)P(i)]-incorporation was significantly increased (141%) in tissue from NIPS treated animals but attenuated (57%) in tissue from estrogen primed animals. Dopamine uptake kinetics were measured in vivo following manipulation of the heterotrimeric G-protein by pertussis toxin (0.5 microg, 48 h). Pertussis toxin significantly inhibited dopamine uptake at all concentrations of dopamine examined. Co-treatment with estrogen and pertussis toxin resulted in a further attenuation of dopamine transport at high but not low dopamine concentrations. These data are consistent with an estrogen mediated alteration of G-protein activity and support the hypothesis that estrogen may alter transporter activity through a modulation of dopamine D2 autoreceptor/G alpha(i/o) protein coupling.