A method for inducible gene over-expression and down-regulation in emerging model species using Pogostick.
Nontraditional model species need new tools for the functional testing of genes, both conserved and lineage-specific genes. These tools should enable the exploration of gene function, either via knock-downs of endogenous genes or via over-expression and ectopic expression of transgenes. We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ-line transformation by the piggyBac transposable element. The vector currently uses the heat-shock promoter Hsp70 from Drosophila melanogaster to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. Here we introduce the main features of Pogostick and how candidate genes can be inserted into the vector for use in either over-expression or down-regulation experiments. In addition, we also test Pogostick in two insect species, D. melanogaster and the emerging model butterfly Bicyclus anynana. We over-express the fluorescent protein DsRed during the larval and pupal stages of D. melanogaster development, and down-regulate DsRed in a line constitutively expressing this gene in the eyes. We then test the over-expression of Ultrabithorax (Ubx) in B. anynana, and obtain sequences flanking the Pogostick genomic insertions. This new vector will allow emerging model species to enter the field of functional genetics with few hurdles.
Institute of Entomology and Molecular Biology, College of Life Sciences, Chongqing Normal University, Chongqing, P. R. China.
Bin
B
Chen
Antónia
A
Monteiro
D000818:Animals; D001483:Base Sequence; D002080:Butterflies / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D015536:Down-Regulation; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D023281:Genomics; D006801:Humans; D055029:Inverted Repeat Sequences; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D030781:Organisms, Genetically Modified; D010957:Plasmids / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; C413663:fluorescent protein 583