Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
AssigneeRegents of the University of Minnesota
InventorsPerry B. Hackett, Karl J. Clark, Zoltan Ivics, Zsuzsanna Izsvak, Scott C. Fahrenkrug
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Abstract
The present invention provides for transposon vectors encoding expression control region-traps and gene-traps. Also provided are dicistronic vectors. Certain embodiments of the invention contain internal ribosome entry sites.
Claims166
A nucleic acid fragment comprising a nucleic acid sequence comprising a coding sequence, the nucleic acid sequence positioned between at least two inverted repeats wherein the inverted repeats can bind to an SB protein.
A method for identifying an enhancer in a cell comprising:
- (a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising a detectable marker coding sequence encoding a detectable marker or a selectable marker, wherein the detectable marker coding sequence is operably linked to a weak promoter, the nucleic acid sequence positioned between at least two inverted repeats, wherein the inverted repeats can bind to an SB protein;
- (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell or its progeny within a domain that contains an enhancer.
A method for identifying a genomic coding sequence in a cell comprising:
- (a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) a splice acceptor site and (iii) an internal ribosome entry site;
- wherein the splice acceptor site and internal ribosome entry site are each operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats, and wherein the inverted repeats can bind to an SB protein;
- and
- (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated within a genomic coding sequence of the cell or its progeny.
A method for identifying the function of an analyte coding sequence comprising:
- (a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween, the internal ribosome entry site being operably linked to the detectable marker coding sequence, the nucleic acid fragment positioned between at least two inverted repeats, and wherein the inverted repeats can bind to an SB protein;
- (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell and that the analyte coding sequence is expressed;
- and
- (c) determining whether a phenotype of the cell or its progeny containing the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment, wherein an altered phenotype indicates that the analyte coding sequence plays a function in the phenotype.
A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising a coding sequence, the nucleic acid sequence positioned between at least two inverted repeats wherein the inverted repeats can bind to an SB protein;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
The gene transfer system of 49 wherein the SB protein has an amino acid sequence identity of least about 80% to SEQ ID NO:1.
A method for producing a transgenic animal comprising:
- (a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising a heterologous coding sequence, the nucleic acid sequence positioned between at least two inverted repeats, wherein the inverted repeats can bind to an SB protein to yield a transgenic cell;
- and
- (b) growing the transgenic cell into a transgenic animal.
The method of 73 wherein the SB protein has an amino acid sequence identity of least about 80% to SEQ ID NO:1.
A gene transfer system to introduce a nucleic acid sequence into the DNA of a fish comprising a nucleic acid fragment comprising a nucleic acid sequence comprising an internal ribosome entry site, wherein the nucleic acid fragment is capable of integrating into the genomic DNA of a fish.
A transgenic fish or fish cell comprising a heterologous internal ribosome entry site.
A nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
A nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
A nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
A method for identifying an enhancer in an isolated or cultured vertebrate cell comprising:
- (a) introducing into an isolated or cultured vertebrate cell
- (i) a nucleic acid fragment comprising a nucleic acid sequence comprising a detectable marker coding sequence encoding a detectable marker or a selectable marker, wherein the detectable marker coding sequence is operably linked to a weak promoter, the nucleic acid sequence positioned between at least two inverted repeats that can bind to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell;
- and
- (ii) a transposase source selected from the group consisting of a transposase and a nucleic acid encoding a transposase, wherein the transposase is an SB protein;
- (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell or its progeny within a domain that contains an enhancer.
A method for identifying a genomic coding sequence in an isolated or cultured vertebrate cell comprising:
- (a) introducing into an isolated or cultured vertebrate cell
- (i) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) a splice acceptor site and (iii) an internal ribosome entry site;
- wherein the splice acceptor site and internal ribosome entry site are each operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats that can bind to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell;
- and
- (ii) a transposase source selected from the group consisting of a transposase and a nucleic acid encoding a transposase, wherein the transposase is an SB protein;
- (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated within a genomic coding sequence of the cell or its progeny.
A method for identifying the function of an analyte coding sequence comprising:
- (a) introducing into an isolated or cultured vertebrate cell
- (i) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween, the internal ribosome entry site being operably linked to the detectable marker coding sequence, the nucleic acid fragment positioned between at least two inverted repeats that can bind to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell;
- and
- (ii) a transposase source selected from the group consisting of a transposase and a nucleic acid encoding a transposase, wherein the transposase is an SB protein;
- (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell and that the analyte coding sequence is expressed;
- and
- (c) determining whether a phenotype of the cell or its progeny containing the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment, wherein an altered phenotype indicates that the analyte coding sequence plays a function in the phenotype.
A nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell, and wherein the weak promoter directs higher expression of the detectable or selectable marker when the nucleic acid fragment integrates into the DNA of the cell near an enhancer than when it integrates elsewhere into the DNA of the cell.
A nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell.
A nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell.
A gene transfer system to introduce a nucleic acid sequence into the DNA of an isolated or cultured vertebrate cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell, and wherein the weak promoter directs higher expression of the detectable or selectable marker when the nucleic acid fragment integrates into the DNA of the cell near an enhancer than when it integrates elsewhere into the DNA of the cell.;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
A gene transfer system to introduce a nucleic acid sequence into the DNA of an isolated or cultured vertebrate cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
A gene transfer system to introduce a nucleic acid sequence into the DNA of an isolated or cultured vertebrate cell comprising:
- (a) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell;
- and
- (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
A gene transfer system to introduce a nucleic acid sequence into the DNA of a fish comprising a nucleic acid fragment comprising a nucleic acid sequence comprising an internal ribosome entry site, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, wherein the nucleic acid fragment is capable of integrating into the genomic DNA of a fish.
A transgenic fish or fish cell comprising a nucleic acid sequence comprising a heterologous internal ribosome entry site, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
Dependencies are inferred from the claim text.