Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac
AssigneeUniversity of Notre Dame du Lac
InventorsMalcolm J. Fraser JR., Xu Li, Teresa Beam, Aurelie Hua-Van
Life of this patent
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Abstract
More efficient transfer of genes into host cells or embryos to transform the cells or embryos is facilitated by transposition vectors using the minimal amount of nucleotide sequences in the transposon piggyBac required for gene transfer. The transformed cells or embryos may be developed into transgenic organisms.
Claims19
A DNA molecule comprising at least 163 consecutive nucleotide base pairs of the 3′ terminal region beginning at the 3′ terminal base pair, and at least 125 consecutive nucleotide base pairs of the 5′ terminal region beginning at the 5′ terminal base pair, of the piggyBac molecule, said region extending from the restriction site SacI to the end of the piggyBac molecule.
A genetic cartridge designated ITR.
A vector designated pXL-Bac as shown in FIG. 3.
A cartridge comprising the minimal piggyBac 5N terminal regions and intervening sequences.
A cartridge comprising the configuration designated [IR/31BP/TR/TTAA . . . 589. . . TT/AA/TR/IR].
A kit for the construction of piggyBac transformation vectors from existing plasmids, said kit comprising the vectors designated A, B, p3E1.2, and a helper construct.
A method of constructing a mobilized and operational transposable piggyBac vector, said method comprising:
- (a) inserting a DNA molecule that contains the sequences essential for piggyBac mobility into a recipient plasmid;
- and
- (b) converting the recipient plasmid into an operational transposable sequence by means of a piggyBac transposon gene or a protein.
A method of transferring a segment of a DNA molecule into a genome of a host cell or embryo, said method comprising:
- (a) obtaining a transposon vector that contains the minimal piggyBac elements and the segment of the DNA molecule;
- (b) transferring the vector into the host cell or embryo;
- (c) co-injecting a helper plasmid expressing a transposase specific for piggyBac elements in the vector;
- and
- (d) providing conditions allowing insertion of the segment into the genome of the host cell or embryo, and excision of piggyBac elements.
A genetic construct designated p32 as shown in FIG. 4.
A method for making a transgenic organism, said method comprising:
- (a) inserting a DNA molecule into a minimal piggyBac construct to be transferred into a recipient cell or embryo;
- (b) transferring the construct into the cell or embryo of the organism under conditions that allow inserting of the DNA molecule into the genome of the recipient cell or embryo;
- and
- (c) allowing the cell or embryo to develop into a transgenic organism.
A plasmid designated pCaSpeR-hs-orf.
A plasmid designated pBSII-IFP2-orf.
Dependencies are inferred from the claim text.