Method of transferring a DNA sequence to a cell in vitro
First Claim
1. A method of transferring a DNA sequence to an oviduct or embryo of an egg laying species, comprising:
- (a) introducing a DNA transfer vector into a cell in vitro to yield a transformed cell which produces recombinant virus, wherein the recombinant virus comprises RNA transcribed from the DNA vector, and wherein the DNA transfer vector comprises linked;
(i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) a DNA sequence comprising at least one open reading frame inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector; and
(vi) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp;
(b) introducing the recombinant virus to the oviduct or embryo of the egg laying species; and
(c) identifying egg laying animals which express the DNA sequence.
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Accused Products
Abstract
Improved recombinant retrotransposon vectors for gene transfer are disclosed. The synthetic vectors are truncated so as to reduce or altogether eliminate homologous recombination with retroviral helper sequences found in helper cells used to propagate the vectors, making them safer for use in humans and providing more space for therapeutic genes. The vectors transmit foreign DNA efficiently, are stable, enable abundant RNA expression from the retrotransposon transcriptional promoter, and through their diversity permit many useful applications in therapeutics and transgenics. Methods are described for rescuing tissue-specifics promoters obtaining expression in primary cells, mapping the genome and other techniques of therapeutic and transgenic utility.
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Citations
10 Claims
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1. A method of transferring a DNA sequence to an oviduct or embryo of an egg laying species, comprising:
-
(a) introducing a DNA transfer vector into a cell in vitro to yield a transformed cell which produces recombinant virus, wherein the recombinant virus comprises RNA transcribed from the DNA vector, and wherein the DNA transfer vector comprises linked;
(i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) a DNA sequence comprising at least one open reading frame inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector; and
(vi) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp;
(b) introducing the recombinant virus to the oviduct or embryo of the egg laying species; and
(c) identifying egg laying animals which express the DNA sequence.
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2. A method of transferring a DNA sequence to an oviduct or embryo of an egg laying species comprising:
-
(a) introducing a DNA transfer vector into the oviduct or embryo of the egg laying species, wherein the DNA transfer vector comprises linked;
(i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) an expression cassette inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector, wherein the expression cassette comprises a DNA sequence comprising at least one open reading frame that is operably linked to a transcription unit; and
(vii) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp; and
(b) identifying an animal of the egg laying species which expresses the DNA sequence.
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3. A method of transferring a DNA sequence to an oviduct of an egg laying species comprising:
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(a) introducing a DNA transfer vector into the oviduct of the egg laying species, wherein the DNA transfer vector comprises an expression cassette comprising a DNA sequence encoding a protein or RNA that is operably linked to a transcription unit, and wherein the expression cassette is inserted into an LTR-containing vector to form the DNA transfer vector; and
(b) identifying an animal of the egg laying species which expresses the protein or RNA encoded by the DNA sequence.
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4. A method of transferring a DNA sequence to a recipient cell comprising:
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(a) introducing a DNA transfer vector into a cell in vitro to yield a transformed host cell which produces recombinant virus, wherein the recombinant virus comprises RNA transcribed from the DNA vector, and wherein the DNA transfer vector comprises linked;
(i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) a DNA sequence comprising at least one open reading frame inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector; and
(vii) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp; and
(b) infecting recipient cells in vitro with the recombinant virus to yield infected recipient cells. - View Dependent Claims (5)
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6. A method of transferring a DNA sequence into a cell comprising:
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introducing a DNA transfer vector into the cell in vitro to yield a transformed cell which produces recombinant virus, wherein the recombinant virus comprises RNA transcribed from the DNA vector, and wherein the DNA transfer vector comprises linked;
(i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) a DNA sequence comprising at least one open reading frame inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector; and
(vii) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp. - View Dependent Claims (7)
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8. A method of transferring a DNA sequence to a mammary cell in vitro comprising:
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(a) introducing a DNA transfer vector into a cell to yield a transformed cell which produces recombinant virus, wherein the recombinant virus comprises RNA transcribed from the DNA vector, and wherein the DNA transfer vector comprises linked;
(i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) an expression cassette inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector, wherein the expression cassette comprises a DNA sequence encoding a protein operably linked to a transcriptional promoter containing a hormone inducible transcription element and a mammary cell specific transcription element; and
(vii) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp; and
(b) introducing the virus into the mammary cell in vitro to yield a transformed mammary cell.
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9. A method of transferring a DNA sequence to a mammary cell comprising:
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introducing a DNA transfer vector into a mammary cell in vitro to yield a transformed mammary cell, wherein the DNA transfer vector comprises (i) a 5′
long terminal repeat (LTR) sequence derived from a retrotransposon comprising a transcription initiation site for RNA;
(ii) an encapsidation sequence positioned 3′
of the 5′
LTR;
(iii) a primer binding site sequence positioned 3′
of the 5′
LTR;
(iv) a 3′
LTR sequence derived from a retrotransposon positioned 3′
of the primer binding site which includes;
(1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′
LTR;
(2) sequences necessary for reverse transcription of the RNA transcript into a double stranded cDNA;
(v) a polypurine tract sequence located 5′
to the 3′
LTR;
(vi) an expression cassette inserted into the vector 3′
of the transcription initiation site in the 5′
LTR of the vector, wherein the expression cassette comprises a DNA sequence encoding a protein operably linked to a transcriptional promoter containing a hormone inducible transcription element and a mammary cell specific transcription element; and
(vii) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell, wherein the vector sequences of (i), (ii), (iii), (iv), and (v) comprise no more than 2 kbp.
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10. A method for preparing genetically modified hematopoietic stem cells derived from bone marrow comprising:
contacting in vitro quiescent bone marrow cells treated with at least one cytokine that induces proliferation of the cells with a recombinant virus so as to yield genetically modified hematopoietic stem cells, wherein the recombinant virus comprises RNA obtained from a retrotransposon vector comprising a DNA sequence comprising at least one open reading frame inserted into the retrotransposon vector 3′
of the transcription initiation site in the 5′
retrotransposon LTR.
Specification