Progress 05/01/04 to 04/30/08
Outputs
OUTPUTS: We have are using an insertional mutagenesis scheme based on remobilization of the piggyback transposon to generate and screen several thousand new insertional mutants in Tribolium castaneum, which is a major coleopteran pest of stored grains. Our international team, combining the expertise of 2 research groups in Germany and 3 American research groups, has screened more than 11,000 lines for new enhancer traps, homozygous lethal and/or sterile mutations. In addition to the new lines being maintained in our labs for further analysis, we have distributed more than 70 interesting lines to more than 20 other research groups. In addition to new enhancer traps, we have analyzed the new insertional mutants for recessive lethality and sterility. Approximately 4% of the new insertional mutations (more than 300) are lethal in homozygous form and 17 are sterile. We have cloned and sequenced 75% of the junction sites from these new insertion lines. Most of them fall within genes (77%).
Almost 50% of the latter map to introns; 25% occur in coding regions; while the remaining 25% are equally distributed between 5' and 3' UTRs. A database containing information regarding insertion sites and the genes affected is being constructed. Insertion sites will also be viewable in the genome browser in Beetlebase. Mapping the insertion sites allowed us to determine the distribution of remobizatian events for piggyBAC in Tribolium. The original donor transposon, on linkage group 3 (LG3) contains an EGFP reporter driven by the 3X3P promoter, which serves as both a transformation marker (expressed in pupal and adult eyes), and as a reporter for new enhancer traps (expressed in novel patterns). Although we documented global reinsertion of this construct, there was a slight perference for local remobilization, estimated to be approximately 3 times more frequent than reinsertion at sites distant from the original location (35 insertions on LG3 versus and average of 11 insertions on
each of the other chromosomes). A new donor transposon, containing the native eye color gene Tc-vermillion (Tc-v) as the transformation marker and inserted on the X chromosome, has also been used to generate new insertion lines. Similar to the original construct, this transposon shows a preference for local transposition events at about the same rate (325 insertions on the X chromosome versus 1008 total insertions on the 9 autosomes). However, since the donor transposon is located on the X chromosome, it is a simple matter to select against these local reinsertion events early in the mutagenesis scheme, if desired.
PARTICIPANTS: Nothing significant to report during this reporting period.
TARGET AUDIENCES: Nothing significant to report during this reporting period.
PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Remobilization of the piggyBAC transposon is a highly efficient method by which to obtain genome-wide, saturation mutagenesis in Tribolium, and perhaps in other economically important insects susceptible to genome transformation by this transposon.
Publications
- Lorenzen, MD, T Kimzey, TD Shippy, SJ Brown, RE Denell, RW Beeman (2007) piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Molecular Biology 16:265-75.
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Progress 01/01/07 to 12/31/07
Outputs
We have are using an insertional mutagenesis scheme based on remobilization of the piggyback transposon to generate and screen several thousand new insertional mutants in Tribolium castaneum, which is a major coleopteran pest of stored grains. Our international team, combining the expertise of 2 research groups in Germany and 3 American research groups, has screened more than 11,000 lines for new enhancer traps, homozygous lethal and/or sterile mutations. In addition to the new lines being maintained in our labs for further analysis, we have distributed more than 70 interesting lines to more than 20 other research groups. In addition to new enhancer traps, we have analyzed the new insertional mutants for recessive lethality and sterility. Approximately 4% of the new insertional mutations (more than 300) are lethal in homozygous form and 17 are sterile. We have cloned and sequenced 75% of the junction sites from these new insertion lines. Most of them fall within genes
(77%). Almost 50% of the latter map to introns; 25% occur in coding regions; while the remaining 25% are equally distributed between 5' and 3' UTRs. A database containing information regarding insertion sites and the genes affected is being constructed. Insertion sites will also be viewable in the genome browser in Beetlebase. Mapping the insertion sites allowed us to determine the distribution of remobizatian events for piggyBAC in Tribolium. The original donor transposon, on linkage group 3 (LG3) contains an EGFP reporter driven by the 3X3P promoter, which serves as both a transformation marker (expressed in pupal and adult eyes), and as a reporter for new enhancer traps (expressed in novel patterns). Although we documented global reinsertion of this construct, there was a slight perference for local remobilization, estimated to be approximately 3 times more frequent than reinsertion at sites distant from the original location (35 insertions on LG3 versus and average of 11
insertions on each of the other chromosomes). A new donor transposon, containing the native eye color gene Tc-vermillion (Tc-v) as the transformation marker and inserted on the X chromosome, has also been used to generate new insertion lines. Similar to the original construct, this transposon shows a preference for local transposition events at about the same rate (325 insertions on the X chromosome versus 1008 total insertions on the 9 autosomes). However, since the donor transposon is located on the X chromosome, it is a simple matter to select against these local reinsertion events early in the mutagenesis scheme, if desired.
Impacts
Remobilization of the piggyBAC transposon is a highly efficient method by which to obtain genome-wide, saturation mutagenesis in Tribolium, and perhaps in other economically important insects susceptible to genome transformation by this transposon.
Publications
- Lorenzen, MD, T Kimzey, TD Shippy, SJ Brown, RE Denell, RW Beeman (2007) piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Molecular Biology 16:265-75.
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Progress 01/01/06 to 12/31/06
Outputs
The goal of this project is to generate and screen several thousand new insertional mutants in the red flour beetle, Tribolium castaneum. Tribolium is a pest of stored grain and an excellent genetic model organism for several highly desctructive beetle species. We are using an insertional mutagenesis scheme based on the piggyBAC transposon. A non-autonomous version of this element carrying EGFP driven by an eye-specific promoter as a transformation marker is remobolized by supplying the piggyBAC transposase. The transposase is supplied by a non-autonomous minos element. We have screened over 8,000 new lines for new enhancer traps, homozygous lethal and/or sterile mutations. We are maintaining over 400 new lines in our labs and have distributed transgenic lines to over 20 other research labs. We are also generating new transposon constructs to optimize the remobilization strategy. The original donor element contains an EGFP reporter driven by the 3X3P promoter which
serves as both a transformation marker when expressed in the eyes, and as a reporter in enhancer trapping when expressed in novel patterns. We have found that the element is remobilized locally and is subject to several CNS specific expression patterns in enhancer traps. These CNS specific expression patterns likely reflect limitations of the 3XP3 promoter. We have developed a new donor transposon that contains a native eye color gene Tc-vermillion (Tc-v) as the transformation marker, in place of EGFP. Transformants are recognized by the rescue of a Tc-v null mutation. This allows for selection of positive individuals without epi-fluorescence microscopy. In addition, it abolishes the fluorescent background to allow for more exact detection of enhancer traps. The endogenous Tribolium hunchback promotor has been included to efficiently identify enhancer traps. We have also included an attP to allow for future modification of the insertion, FRT sites to allow for the directed generation
of balancers by specific genomic recombination, and an I-Sce1 restriction site to allow for generation of additional alleles by cut and repair of adjacent DNA. A transgenic line containing this construct inserted on the X chromosome (XL-1) has been generated and is currently being used in the mutagenesis scheme. This is an important innovation to easily select against local remobilization. We are also constructing a database to provide community access to information regarding these new mutants.
Impacts
In this project we are identifying new genes and regulatory elements by the remobilization of the piggyBAC transposon. Knowledge gained regarding these genes and regulatory regions can be exploited to develop new strategies in pest management for several related beetle species that are highly destructive to crops and forests
Publications
- Lorenzen, MD, T Kimzey, TD Shippy, SJ Brown, RE Denell, RW Beeman (2006) piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Molecular Biology in press.
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Progress 01/01/05 to 12/31/05
Outputs
The goal of this project is to saturate the genome of the red flour beetle, Tribolium castaneum with insertional mutations. This project is a collaboration between two research groups in Germany and three research groups in Manhattan, KS. We held our second international meeting in Gottingen, Germany. As part of our goal to analyze 15,000 new insertion lines, each lab has over 2000 lines in progess using jumpstarter systems based on the piggyBac tranpson. A new jumpstarted system based on the Minos transposon has been constructed and is being tested for use in the following year. A manuscript describing the pilot experiment leading to this project is being completed for submission to the journal Genetics.
Impacts
We have identified several new insertional mutations that are lethal in the homozygous state. These mutations identify genes that may be targets for new pest management strategies in beetles.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The goal of this project is to saturate the genome of the red flour beetle, Tribolium castaneum with insertional mutations using the piggyback transposon. This project is a collaboration between two research groups in Germany and three research groups in Manhattan, KS. At our first international meeting (held in Manhattan, KS) we finalized the first mutagenesis protocols and nomenclature. In the pilot screen of 411 mutant lines we found 19 homozygous lethalmutations, 1 male sterile mutation and 27 new pupal enhancer traps. We are investigating the lethal phenotype of the 19 recessive lethal mutations.
Impacts
We are using a functional genomic approach based on insertional mutagenesis to directly link DNA sequence information to biological and ecological functions. Genes discovered in this work may provide insight into resistance mechanisms, biosynthetic pathways and developmentally important genes that will provide new potential targets for the development of novel pest management strategies relevant to Tribolium and related pest species.
Publications
- No publications reported this period
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