Insulated piggyBac vectors for insect transgenesis

BACKGROUND: Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells. RESULTS: To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. CONCLUSION: The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species

Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

<p>Abstract</p> <p>Background</p> <p>In male <it>Drosophila melanogaster</it>, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.</p> <p>Results</p> <p>MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in <it>Drosophila</it>. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity <it>in vitro </it>and UAS-DsRed activation in <it>Drosophila</it>. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-<it>lacZ </it>and UAS-<it>arm-lacZ </it>reporter genes. The latter utilizes the constitutive promoter from the <it>arm </it>gene to drive <it>lacZ </it>expression. In contrast to the strong induction of UAS-DsRed expression, UAS-<it>arm-lacZ </it>expression increased by about 2-fold in both sexes.</p> <p>Conclusions</p> <p>Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-<it>DsRed </it>but not UAS-<it>arm-lacZ </it>genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.</p

Abnormal Dosage Compensation of Reporter Genes Driven by the Drosophila Glass Multiple Reporter (GMR) Enhancer-Promoter

In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3′ end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly

<div><p>In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly <i>Drosophila melanogaster</i>, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly <i>Lucilia cuprina</i> have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in <i>D</i>. <i>melanogaster</i>. Recent evidence suggests that the <i>D</i>. <i>melanogaster</i> fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in <i>Drosophila</i> species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in <i>L</i>. <i>cuprina</i>. The other two Muller element F genes were found to be autosomal in <i>L</i>. <i>cuprina</i>, whereas two Muller element B genes were found on the same region of the X chromosome as the <i>L</i>. <i>cuprina</i> orthologs of the <i>D</i>. <i>melanogaster Ephrin</i> and <i>gawky</i> genes. We find that the <i>L</i>. <i>cuprina</i> X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in <i>Drosophila</i>, it appears that the <i>Lucilia</i> dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.</p></div

Relative abundance of candidate X-linked genes and known X-linked transgenes in male and female genomic DNA.

<p>^a,b,cSuperscript indicates strain that was the source of genomic DNA for analysis.</p><p>^a: wild type,</p><p>^b: SLAM5, and</p><p>^c: DR3-9.</p><p>^d. For each gene the male sample was chosen as the control/ calibrator sample and set to equal 1.0.</p><p>^e. Two <i>p</i>-values for each gene were calculated from the competing hypotheses that either the Male-Female/2 difference was zero or that the Male-female difference was zero. The latter would be expected for a M/F ratio of one.</p><p>Relative abundance of candidate X-linked genes and known X-linked transgenes in male and female genomic DNA.</p

Dosage compensation of X chromosome genes in <i>L</i>. <i>cuprina</i>

<p>^a. Mean of 3 independent experiments. Expression was normalized to the 28S rRNA reference gene.</p><p>^b. Student's t-tests were performed for each gene separately</p><p>^c. With the exception of <i>Lcaru</i>, all genes are orthologs of genes located on the fourth chromosome in <i>D</i>. <i>melanogaster</i>. The <i>D</i>. <i>melanogaster aru</i> gene is located on chromosome 2.</p><p>Dosage compensation of X chromosome genes in <i>L</i>. <i>cuprina</i></p

X-linked transgenes are not fully dosage compensated in <i>L</i>. <i>cuprina</i>.

<p>qRT-PCR of marker gene (ZsGreen or DsRed-express2) RNA in hemisected adult males and females from the transgenic lines SLAM5, EF 1–2 or DR3-9. All lines carry a single copy of the marker gene on the X chromosome. Transcript levels were normalized to 28S rRNA. As expression of the marker genes was driven by the constitutive promoter from the autosomal <i>Lchsp83</i> gene, <i>Lchsp83</i> transcript levels are shown for comparison. Mean female/male ratio +/- standard error from three biologically independent replicate experiments are shown.</p

Schematic illustration of male metaphase chromosomes in <i>D</i>. <i>melanogaster</i> and <i>L</i>. <i>cuprina</i>.

<p>Muller elements (A-F) are indicated. The chromosomal location of linkage group F genes in <i>L</i>. <i>cuprina</i> was unclear from earlier genetic analysis but it has been suggested that these genes are on the X chromosome (this uncertainty is indicated by a question mark). In <i>L</i>. <i>cuprina</i>, C-banding produces dark staining of the sex chromosomes, except for a lighter staining region in the distal portion of the long arm of the X chromosome that is thought to contain active genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141544#pone.0141544.ref013" target="_blank">13</a>]. While the X chromosome is the largest chromosome, it is not drawn to scale so as to highlight the C-banding pattern.</p

<i>L</i>. <i>cuprina</i> larvae with X-linked or autosomal fluorescent protein transgenes.

<p>The lines carry a constitutively expressed DsRed-express2 marker gene. Larvae from an X-linked line (SLAM5) and an autosomal line (EF3C) are shown. In the X-linked line, the most brightly fluorescent larvae develop as females (F). The more weakly fluorescent larvae develop as males (M). In contrast, larvae from the EF3C line show a uniform level of fluorescence. Two representative larvae from this line are shown, the sex of the larvae is unknown.</p