Biomedical subjects

A M Handler

Publications and source records attributed to A M Handler.

At least 19 recordsLinked to original sources

PubMed2005

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

Animals↗

PubMed2004

piggyBac transformation of the New World screwworm, Cochliomyia hominivorax, produces multiple distinct mutant strains.

Sterile insect technique (SIT) programs are designed to eradicate pest species by releasing mass-reared, sterile insects into an infested area. The first major implementation of SIT was the New World Screwworm Eradication Program, which successfully eliminated the New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), from the Continental US, Mexico and much of Central America. Ionizing radiation is currently used for sterilization, but transgenic insect techniques could replace this method, providing a safer, more cost-effective alternative. Genetic transformation methods have been demonstrated in NWS, and verified by Southern blot hybridization, PCR and sequencing of element insertion junctions. A lethal insertional mutation and enhancer detection-like phenotypic expression variations are presented and discussed. In addition to supporting the eradication efforts, transformation methods offer potential means to identify genes and examine gene function in NWS.

Animals↗

PubMed2002

Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient.

Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac-mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.

Animals↗

PubMed2001

Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP.

Germ-line transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species.

Animals↗

PubMed2001

A current perspective on insect gene transformation.

The genetic transformation of non-drosophilid insects is now possible with several systems, with germ-line transformation reported in published and unpublished accounts for about 12 species using four different transposon vectors. For some of these species, transformation can now be considered routine. Other vector systems include viruses and bacterial symbionts that have demonstrated utility in species and applications requiring transient expression, and for some, the potential exists for genomic integration. Many of these findings are quite recent, presenting a dramatic turning point in our ability to study and manipulate agriculturally and medically important insects. This review discusses these findings from the perspective of all the contributions that has made this technology a reality, the research that has yet to be done for its safe and efficient use in a broader range of species, and an overview of the available methodology to effectively utilize these systems.

Animals↗

PubMed2001

Polyubiquitin-regulated DsRed marker for transgenic insects.

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP The DsRed fluorescentprotein has spectral characteristics that are most distinct from GFP and GFP variants, and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitin-regulated DsRed1 were detected throughout development at a relatively high frequency, and they exhibited brighter fluorescence than transformants marked with EGFP. The use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressedfrom a reporter construct could be unambiguously detected when co-expressed with EGFP DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.

Animals↗

PubMed2000

The piggyBac transposon mediates germ-line transformation in the Oriental fruit fly and closely related elements exist in its genome.

Germ-line transformation of a white eye strain of the Oriental fruit fly, Bactrocera dorsalis, was achieved with the piggyBac vector, derived from a transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The vector was marked with the medfly white+ gene cDNA, and three transgenic lines were identified at a frequency of approximately 2% per fertile G0. Vector integrations were verified by Southern DNA hybridization, which also revealed the presence of endogenous genomic elements closely related to piggyBac. Approximately 10-20 elements per genome were evident in several B. dorsalis strains, and sequence analysis of 1.5 kb gene amplification products from two wild strains and the white eye host strain indicated 95% nucleotide and 92% amino acid sequence identity among resident elements and the T. ni element. PiggyBac was not evident by hybridization in other tephritid species, or insects previously transformed with the transposon. This is the first discovery of piggyBac beyond T. ni, and its existence in a distantly related species has important implications for the practical use of the vector and insects transformed with it.

Amino Acid Sequence↗

PubMed1999

Germline transformation of Drosophila melanogaster with the piggyBac transposon vector.

Germline transformation of Drosophila melanogaster was attempted with the piggyBac gene-transfer system from the cabbage looper moth, Trichoplusia ni. Using a self-regulated transposase helper and a white marked vector, a transformation frequency of 1-3% per fertile G0 was obtained, similar to that previously achieved in the medfly. Use of an hsp70-regulated helper increased this frequency more than eight-fold. Transformation with a vector marked with white and green fluorescent protein (GFP) under polyubiquitin-nuclear localizing sequence regulation yielded seventy G1 transformants which all expressed GFP, but only twenty-seven of these expressed eye pigmentation that would have allowed their selection based on white+ expression. PiggyBac transformation in two distantly related dipteran species and efficient expression of the gfp marker supports the potential use of this system in other dipterans, and perhaps insects in general.

Animals↗

PubMed1998

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly.

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata. A piggyBac vector marked with the medfly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3-5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G1 transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a piggyBac-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic piggyBac TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the piggyBac system will be functional in a broad range of insects.

Animals↗

PubMed1997

A new hobo, Ac, Tam3 transposable element, hopper, from Bactrocera dorsalis is distantly related to hobo and Ac.

A new transposable element from the hobo, Ac, Tam3 transposon family was isolated as a genomic clone from the oriental fruit fly, Bactrocera dorsalis. It is approximately 3.1 kb in length with 19-bp inverted terminal repeat sequences having a single mismatch. Though sharing several amino acid sequence identities with other hAT elements, it is distantly related to both hobo and Ac. Among hAT elements thus far described in insects, it is apparently the most distantly related to hobo.

Amino Acid Sequence↗

PubMed1997

Developmental regulation of yolk protein gene expression in Anastrepha suspensa.

A partial cDNA clone for the 48,000 dalton yolk polypeptide gene from Anastrepha suspensa was isolated from a cDNA expression library using a yolk polypeptide antibody probe and hybridization to the Drosophila melanogaster yolk protein 1 gene. The sequenced DNA has greatest homology to the yolk protein genes from Ceratitis capitata, D. melanogaster, and Calliphora erythrocephala and, similar to these genes, shares amino acid sequence domains with those from lipases. RNA hybridization studies indicated that the yolk protein gene expression is completely female-specific and limited to the ovaries, without apparent regulation by 20-hydroxyecdysone or juvenile hormone. This is in contrast to an earlier study which suggested, based on immunological probes, that a very low level of yolk protein synthesis occurred in fat body and was not sex-specific.

Amino Acid Sequence↗

PubMed1997

P element excision in Drosophila is stimulated by gamma-irradiation in transient embryonic assays.

The influence of gamma-irradiation on P element excision and excision-site repair mechanisms was directly tested by embryonic somatic excision assays. Preblastoderm P[ry+, delta 2-3](99B) embryos, having a stable source of somatically active P transposase, were irradiated previous to injection with P excision indicator plasmids. Frequencies of precise or nearly precise P excision increased with gamma-ray doses ranging from 0.5 to 3.5 Gy. Higher doses resulted in frequencies close to that in unirradiated embryos, though considerable embryonic lethality was also evident at these doses. A direct positive interaction between gamma-irradiation and P element activity is concluded.

Animals↗

PubMed1997

A Drosophila melanogaster hobo-white(+) vector mediates low frequency gene transfer in D. virilis with full interspecific white(+) complementation.

Transformation of a Drosophila virilis white mutant host strain was attempted using a hobo vector containing the D. melanogaster mini-white(+) cassette (H[w(+), hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (HFL1), one containing only the white(+) marked vector, and a sibling line containing the vector as well as an HFL1 helper integration. An approximate total transformation frequency of 1% is deduced. A high frequency of wing and eye morphology mutants were also observed, suggesting that hobo may have mobilized a related element in D. virilis. The data reaffirms a relatively low transformation vector activity for the hobo transposon in D. virilis; however, nearly full interspecific expression white(+) marker supports its possible function in other species as well.

ATP-Binding Cassette Transporters↗

PubMed1996

The hobo transposable element excises and has related elements in tephritid species.

Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.

Amino Acid Sequence↗

PubMed1995

The hobo transposable element has transposase-dependent and -independent excision activity in drosophilid species.

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry42) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo, hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry42 and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.

Animals↗

PubMed1993

A functional analysis of the P-element gene-transfer vector in insects.

A P-element mobility excision assay was used to determine if non-drosophilid insects could support P gene vector function. Present studies included the testing of Muscids, Sphaerocerids, and Phorids, none of which were able to support P mobility. A new excision indicator plasmid was developed allowing the detection and recovery of virtually all P-element excision products. The frequency and sequence analysis of excision products from Drosophila melanogaster and another drosophilid, Chymomyza procnemis, indicated both quantitative and qualitative differences in the activity of transposase. The quantitative relationships observed in the original assay were maintained, and qualitative differences in transposase activity were reflected in the sequence of the empty donor sites. The results suggest that host factors are involved in cutting and ligating P-element DNA during excision, with transposase facilitating these processes. Possible limitations on P mobility by abnormal transposase transcript processing were tested in Anastrepha suspensa using transposase-encoding plasmids having deleted intron sequences. A transposase cDNA supported normal P excision in D. melanogaster, and a low level of mobility in A. suspensa. Possible applications of gene transfer in insects are presented, in particular methods to genetically sterilize and sex insects for the sterile-insect technique.

Animals↗

PubMed1993

Negative regulation of P element excision by the somatic product and terminal sequences of P in Drosophila melanogaster.

A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phs pi) effectively repressed P excision in a dose-dependent manner at very low concentrations relative to somatically active transposase (encoded by the hs pi delta 2-3 gene). Maximum repression required transcription of the complete transposase gene. Dose-dependent repression of P excision was also observed in the presence of a vector plasmid (pCarnegie4) having only the terminal sequences, including transposase binding sites, of the P element. However, repression required considerably higher concentrations of pCarnegie4 than phs pi, and elimination of P excision was not observed.

Animals↗

PubMed1991

P element excision in Drosophila melanogaster and related drosophilids.

The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species. Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlike previous assays, this mobility assay permitted the recovery of excision products from plasmids regardless of whether the excision event was precise or imprecise. Both quantitative and qualitative differences between the products of excision in the various species studied were observed. The frequency with which P element excision products were recovered from D. melanogaster was 10-fold greater than from D. virilis and C. procnemis; however, the proportion of all excision events resulting in the reversion of a P-induced mutant phenotype was the same. Virtually all excision products recovered, including those resulting in a reversion of the mutant phenotype, did not result in the exact restoration of the original target sequence. Sequence analysis suggested that duplex cleavage at the 3' and 5' termini of the P element, or their subsequent modification, occurred asymmetrically and interdependently. P element-encoded transposase was not absolutely required for P element excision.

Animals↗