Controlled activation of non-LTR retrotransposons in mammals
The invention relates to nucleic acids, vector constructs which allow the controlled activation and inhibition of retrotransposition of non-LTR retrotransposons. The methods of this invention are useful for preparing said nucleic acids and vector constructs and introducing them into cells.
1. A nucleic acid molecule comprising:
an autonomous non-long terminal repeat (LTR) retrotransposon that lacks an intact endogenous promoter; and an operably linked inducible first promoter exogenous to the retrotransposon.
2. The nucleic acid molecule of claim 1 , wherein the first promoter comprises a tetracycline-response element (TRE) operably linked to a TRE-responsive promoter.
3. The nucleic acid molecule of claim 2 , wherein the TRE-responsive promoter is a cytomegalovirus promoter.
4. The nucleic acid molecule of claim 1 , wherein the retrotransposon comprises a heterologous gene selected from the group consisting of a reporter gene, a therapeutic gene, and a selectable marker gene.
5. The nucleic acid molecule of claim 4 , wherein the reporter gene is selected from the group consisting of a green fluorescent protein (GFP) gene, an enhanced-GFP (EGFP) gene, and a beta-galactose gene.
6. The nucleic acid molecule of claim 4 , wherein the selectable marker gene confers resistance to an antibiotic.
7. The nucleic acid molecule of claim 6 , wherein the antibiotic is selected from the group consisting of blasticidin and neomycin.
8. The nucleic acid molecule of claim 4 , wherein the heterologous gene is operably linked to a second promoter.
9. The nucleic acid molecule of claim 8 , wherein the first and second promoters are not the same promoter.
10. The nucleic acid molecule of claim 8 , wherein at least one of the first and second promoters is selected from the group consisting of a tissue-specific promoter and a cell-type specific promoter.
11. The nucleic acid molecule of claim 8 , wherein the second promoter is an inducible promoter.
12. The nucleic acid molecule of claim 1 , wherein the retrotransposon comprises a mammalian retrotransposon ORF1, ORF2, 3′ UTR, and poly A signal.
13. The nucleic acid molecule of claim 12 , wherein the mammal is selected from the group consisting of a human, a rat, and a mouse.
14. The nucleic acid of claim 12 , wherein the retrotransposon is a mammalian long interspersed element 1 (LINE-1) type retrotransposon.
15. The nucleic acid molecule of claim 14 , wherein the LINE-1 is codon optimized.
16. The nucleic acid molecule of claim 12 , comprising a heterologous gene in the 3′ UTR, the heterologous gene being selected from the group consisting of a reporter gene, a therapeutic gene, and a first selectable marker gene.
17. The nucleic acid molecule of claim 16 , wherein the reporter gene is selected from the group consisting of a green fluorescent protein (GFP) gene, an enhanced-GFP (EGFP) gene, and a beta-galactose gene.
18. The nucleic acid molecule of claim 16 , wherein the selectable marker gene confers resistance to an antibiotic.
19. The nucleic acid molecule of claim 16 wherein the heterologous gene in the 3 ′ UTR can be transcribed only after retrotransposition of the retrotransposon.
20. The nucleic acid molecule of claim 16 , wherein the heterologous gene is operably linked to a second promoter.
21. The nucleic acid molecule of claim 20 , wherein the first and second promoters are not the same promoter.
22. The nucleic acid molecule of claim 20 , wherein at least one of the first and second promoters is selected from the group consisting of a tissue-specific promoter and a cell-type specific promoter.
23. The nucleic acid molecule of claim 1 , wherein the molecule is a vector construct selected from the group consisting of a viral vector and a plasmid.
24. The nucleic acid molecule of claim 20 , wherein the heterologous gene is positioned in the retrotransposon 3′ UTR such that it can be transcribed only after retrotransposition of the retrotransposon into a cell genome and wherein the molecule further comprises, outside of the retrotransposon, a second selectable marker gene.
25. A method for transfecting mammalian cells comprising the steps of: transfecting into a mammalian cell that expresses a reverse tetracycline-controlled transactivator-protein (rtTA) a nucleic acid molecule that comprises:
an autonomous non-long terminal repeat (LTR) retrotransposon having a mammalian retrotransposon ORF1, ORF2, 3′ UTR and poly A signal but lacking an active endogenous promoter, an inducible first promoter exogenous to and operably linked to the retrotransposon, a first selectable marker gene exogenous to the retrotransposon, and a second selectable marker gene provided in the 3′ UTR and operably linked to a second promoter; exposing the transfected cells to doxycycline and a first selection agent that positively selects cells expressing the first selectable marker, for a time sufficient to select the cells expressing the first selectable marker;
exposing the selected cells to a second selection agent that positively selects cells expressing the second selectable marker, for a time sufficient to select the cells expressing the second selectable marker; and
recovering the cells selected in step (c).
26. The method of claim 25 , wherein the mammalian cells are of human origin.