With the completion of genome sequences of major model organisms, increasingly sophisticated genetic tools are necessary for investigating the complex and coordinated functions of genes. Here we describe a genetic manipulation system termed ''genomic engineering'' in Drosophila. Genomic engineering is a 2-step process that combines the ends-out (replacement) gene targeting with phage integrase C31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knockout line is generated by deleting the target gene and replacing it with an integration site of C31. Second, DNA integration by C31 is used to reintroduce modified target-gene DNA into the native locus in the founder knock-out line. Genomic engineering permits directed and highly efficient modifications of a chosen genomic locus into virtually any desired mutant allele. We have successfully applied the genomic engineering scheme on 6 different genes and have generated at their loci more than 70 unique alleles.cell polarity ͉ ends-out targeting ͉ homologous recombination ͉ phiC31 integrase T he development of homologous recombination (HR)-based gene targeting was a major breakthrough in Drosophila genetics (1, 2). At present, in Drosophila as well as in mice, a HR-based approach is virtually the only way to make directed modifications of a target gene (3, 4). However, because the entire targeting process must be repeated for making each allele, the amount of time and labor may become impractical to make more than just a few targeted alleles. In addition, because of the requirement of HR, it can be very difficult to introduce appreciably complicated DNA sequence modifications by gene targeting. The current lack of adequate genetic tools for directed and efficient modifications of the genome presents a major hurdle in Drosophila genetics today. For example, many of the protein pathways that are highly conserved between Drosophila and vertebrates, such as the cell polarity pathway (5), appear to be exceedingly complex. Rigorous genetic dissections of such intricate protein networks can be highly challenging, because in most cases the functions of mutated or modified individual genes of such pathways can only be assayed by artificial over-expression methods, which often lack the requisite controllability and fidelity of gene expression. One ideal solution would be for each protein gene of interest to generate, at the gene's native genomic locus, a set of defined mutant alleles that are strategically designed to test hypotheses about the protein's in vivo functions and interactions. Furthermore, being able to generate any conceivable alleles of a target gene, such as functional fusion alleles of fluorescent proteins/purification tags or alleles with conditional activities, would also offer us unprecedented freedom and opportunities to explore unique experiments of imaging, proteomics, and disease models.To achieve the goal of such directed, efficient, and versatile modifications of the Drosophila genome, we have developed a...

In this report, we describe several approaches to improve the scalability and throughput of major genetic crosses in ends-out gene targeting. We generated new sets of targeting vectors and fly stocks and introduced a novel negative selection marker that drastically reduced the frequency of false-positive targeting candidates.

Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3’UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.

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This research investigated the function of envelope protein P74 of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in primary infection to host. A p74-inactivation recombinant baculovirus, rAc-gfp(Delta) p74, was constructed by inserting gfp driven by AcMNPV polyhedrin promoter into the p74 locus of AcMNPV genome. Bioassays showed that the P74-null occlusion bodies (OBs) failed to infect its natural host larvae, Spodoptera exigua, per os, while the p74-null budded virus (BVs) could infect host larvae by injection. However, its inability for oral infectivity was rescued by a mixed infection with wild-type OBs or with the purified P74 protein expressed in Spodoptera frugiperda Sf-9 cells, and the P74 protein rescue was in a dosage-dependent manner. The 50% lethal dosage (LD50) value of a P74 overexpression recombinant virus, rAc-p74(++)-polh+, which contained two copies of p74 gene, was not significantly different from that of wild-type virus. One-step growth curve assays of viruses suggested that BV production from cells infected with p74-null virus was similar to that from cells infected with wild-type virus or the P74 overexpression virus. ELISA analysis indicated that P74 protein could bind its host brush border membrane vesicles (BBMV) efficiently with saturation, but it could only bind its sensitive midgut BBMV specifically. In vitro pull-down assay showed that a protein of approximately 35 kDa in the BBMV was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of occlusion-derived virus (ODV) and plays a role in midgut attachment and fusion.

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Perrimon, 1996 SUMMARYPatj has been characterized as one of the so-called polarity proteins that play essential and conserved roles in regulating cell polarity in many different cell types. Studies of Drosophila and mammalian cells suggest that Patj is required for the apical polarity protein complex Crumbs-Stardust (Pals1 or Mpp5 in mammalian cells) to establish apical-basal polarity. However, owing to the lack of suitable genetic mutants, the exact in vivo function of Patj in regulating apical-basal polarity and development remains to be elucidated. Here, we generated molecularly defined null mutants of Drosophila Patj (dPatj). Our data show conclusively that dPatj only plays supporting and non-essential roles in regulating apical-basal polarity, although such a supporting role may become crucial in cells such as photoreceptors that undergo complex cellular morphogenesis. In addition, our results confirm that dPatj possesses an as yet unidentified function that is essential for pupal development.

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a “founder” knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.