Systems for gene targeting and producing stable genomic transgene insertions

a technology of gene targeting and stable insertion, applied in the field of gene targeting and producing stable genomic transgene insertion, to achieve the effect of facilitating the stabilization process and enhancing the efficiency of cassette exchang

Inactive Publication Date: 2009-03-26
HORN CARSTEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0013]The second embodiment disclosed has been termed “conditional excision-competent transformation vectors” (FIG. 4). This embodiment comprises a modified excision-competent transformation vector that contains a transposonR2 half-side in an inverted orientation, relative to the R1 half side, with R2 also flanked by recombinase target sites in inverted orientation. In this configuration, only the TransposonL1 and R1 half-sides can integrate by transposition, and remobilization of the TransposonL1 and R2 half-sides can only occur after a recombinase-mediated inversion between the recombinase target sites. This modification will facilitate the stabilization process, by transposon L1 and R2 half-side deletion, for those excision-competent transformation vectors and / or host species where the primary transposition is highly favored or limited to the internal TransposonL1 and R2 half-sides if R2 was in a normal orientation.

Problems solved by technology

The challenge is to develop a transformation method that prevents re-mobilization of transgenes which have been incorporated into the genome.

Method used

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  • Systems for gene targeting and producing stable genomic transgene insertions
  • Systems for gene targeting and producing stable genomic transgene insertions
  • Systems for gene targeting and producing stable genomic transgene insertions

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Experimental program
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embodiment 1

Excision-Competent Stabilization Vectors

[0029]The experimental steps for the method are described in FIG. 1, and the structure of the excision competent transformation vector, pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 }, is described in FIG. 2. Integration and re-mobilization of the vector was verified by PCR and sequence analysis described in FIG. 3. pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} was constructed based on the transposable element “piggyBac” (see U.S. Pat. No. 6,218,185, the contents of which are incorporated herein by reference). Conventional piggyBac-based transformation vectors (see WO 01 / 14537 and WO 01 / 12667, the contents of which are incorporated herein by reference) typically contain piggyBac-half sides or parts thereof, including 5′ piggyBac terminal sequences (referred to as piggyBacL) and 3′ piggyBac terminal sequences (referred to as piggyBacR), which flank a transformation marker gene and a cloning site to insert the genes-of-interest. (see Handler, A. M., 2001. A current pe...

embodiment 2

Conditional Excision-Competent Transformation Vectors

[0036]The structure of the conditional excision-competent transformation vector, pBac_STBL, as well as the experimental steps are depicted schematically in FIGS. 4 and 6. pBac_STBL is based on the transposable element “piggybac” (see U.S. Pat. No. 6,218,185, the contents of which are incorporated herein by reference) and is a modified version of pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 }. In pBac-STBL the internal transposon half-side (R2) is a duplication of the piggyBac 3′-end, and it is in reverse, or opposite, orientation to R1. In addition, it is flanked in upstream and downstream positions by FRT (FLP recombinase target) sites in opposite directions that create an inversion by recombination in the presence of FLP recombinase (see FIGS. 4 and 6). Therefore, in this vector, only the piggyBacL1 and R1 half sides and intervening DNA can integrate, but re-mobilization of piggyBacR2 together with piggyBacL1 or piggyBacR1 should not be po...

embodiment 3

RMCE with Subsequent Transposon Deletion

[0054]The RMCE-acceptor plasmid, pBac{3×P3-FRT-ECFP-linotte-FRT3} (FIG. 8), is a piggyBac-based transformation vector that was provided additionally with a DNA exchange cassette. This cassette consists of two heterospecific FRT sites (referred to as FRT and FRT3 equivalent to F and F3 (published in European Patent No. EP 0 939 120 A1, the contents of which are incorporated herein by reference)) in parallel orientation.

[0055]European Patent No. EP 0 939 120 A1 (see page 2, line 50 to page 3, line 6) teaches the technology of the RMCE reaction:[0056]“Recombinases such as FLP and Cre have emerged as powerful tools to manipulate the eucaryotic genome (Kilby, N. J., Snaith, M. R., Murray, J. A. H. (1993). Site-specific recombinases: tools for genome engineering. Trends Genet. 9, 413-421, and Sauer B. (1994). Site-specific recombination: developments and applications. Curr. Opin. Biotechnol. 5, 521-527, the contents of which are incorporated by refe...

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Abstract

The novel germ-line transformation systems disclosed in this patent application allow the physical deletion of transposon DNA following the transformation process, and the targeting of transgene integrations into predefined target sites. In this way, transposase-mediated mobilization of genes-of-interest is excluded mechanistically and random genomic integrations eliminated. In contrast to conventional germ-line transformation technology, our systems provide enhanced stability to the transgene insertion. Furthermore, DNA sequences required for the transgene modification (e.g. transformation marker genes, transposase or recombinase target sites), are largely removed from the genome after the final transgene insertion, thereby eliminating the possibility for instability generated by these processes. The RMCE technology, which is disclosed in this patent application for invertebrate organisms (exemplified in Drosophila melanogaster) represents an extremely versatile tool with application potential far beyond the goal of transgene immobilization. RMCE makes possible the targeted integration of DNA cassettes into a specific genomic loci that are pre-defined by the integration of the RMCE acceptor plasmid. The loci can be characterized prior to a targeting experiment allowing optimal integration sites to be pre-selected for specific applications, and allowing selection of host strains with optimal fitness. In addition, multiple cassette exchange reactions can be performed in a repetitive way where an acceptor cassette can be repetitively exchanged by multiple donor cassettes. In this way several different transgenes can be placed precisely at the same genomic locus, allowing, for the first time, the ability to eliminate genomic positional effects and to comparatively study the biological effects of different transgenes.

Description

FIELD OF THE INVENTION[0001]The invention relates to novel methods and techniques to produce transgenic, or genetically modified, organisms (transgenesis). The focus of the innovation is on manipulation techniques that allow for the targeting and the stable anchoring of homologous or heterologous DNA-sequences (in the following description referred to as: “transgene” or “gene-of-interest”) into the genome of a target species. To achieve this goal, we have developed three different systems of transformation vectors that are capable of integrating a transgene into invertebrate and vertebrate organisms via transposon- or recombinase-mediated transformation events. In addition, following the germline transformation procedure, both systems make possible the physical deletion of mobile DNA-sequences, brought in with the vector, from the target genome and therefore to stabilize the gene-of-interest. Stable (genomic) transgene insertions are regarded to be an essential pre-requisite for the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12N15/87
CPCA01K67/0339A01K2217/05C12N2800/90C12N15/8509C12N15/90A01K2227/706
Inventor HORN, CARSTENHANDLER, ALFRED M.
Owner HORN CARSTEN
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