Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-08-10
2001-09-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S461000, C435S462000, C435S463000
Reexamination Certificate
active
06294385
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Efficient insertion of exogenous nucleic acid into the chromosomal and extra-chromosomal nucleic acid of cells is desired in the art of molecular biology to identify chromosomal regions involved in expressing or regulating expression of peptides and proteins or to insert a nucleotide sequence of interest such as a primer binding site into a polynucleotide. This same technology is also advantageously used in developing new therapeutic and pharmacologic agents.
One common method relies upon in vivo Tn5 mutagenesis to insert polynucleotides of interest into cellular DNA and to construct libraries of cells that contain inserted polynucleotides at random or quasi-random locations. Existing in vivo Tn5 mutagenesis methods require target cells to encode transposase, either natively or from an introduced expression construct. Accordingly, it can be necessary to construct a suitable expression system appropriate to each target cell type. This can be time consuming, and requires extensive knowledge of the requirements of each target cell type.
In many cases, the gene that encodes transposase is encoded by an active transposon, which can continue to transpose in a target cell after the initial desired mutagenesis step. Such undesired residual transposition is undesired in that it complicates the analysis of insertional mutant libraries.
Furthermore, many techniques for in vivo Tn5 mutagenesis rely upon a complex biological mechanism for introducing exogenous DNA into the target cells, such as bacteriophage lambda transducing phage or a conjugating plasmid. It would be desirable to avoid requiring such complex biological systems.
The nature of natural Mu DNA, its ends, and its role in transposition are known. The left end includes 3 att repeat sites, denoted L1, L2 and L3 (all parallel). At the left end, only L1 is involved in transpososome formation. The nucleotide sequence of L1 is 5′-TGTATTGATTCACTTGAAGTACGAAAAAAA-3′ (SEQ ID NO:1). L2 and L3 are spaced significantly apart from L1. The right end includes att repeat sites R1, R2 and R3. R1 and R2 are located close to one another and are involved in complex formation. The nucleotide sequence of R1 is 5′-TGAAGCGGCGCACGAAAAACGCGAAAGCGT-3′ (SEQ ID NO:2). The nucleotide sequence of R2is 5′-GAAAGCGTTTCACGATAAATGCGAAAACTT-3′ (SEQ ID NO:3). R3 and L1 are inverted relative to R1 and R2. MuA transposase, encoded by phage Mu, can bind to all 6 att repeat sites, but the MuA tetramer in a transpososome footprints on only L1, R
1
and R2.
The Mu transposition reaction is most efficient when the Mu DNA includes one left end (containing L1, L2 and L3) and one right end (containing R1, R2 and R3). If the transposon ends are precut, strand transfer is most efficient if two right ends (containing R1 and R2) are used. If the non-transferred strand has a few (1 to 16 are equally effective) extra bases at the 5′ end, then the reaction is even more efficient.
A commercial system for inserting a selectable artificial Mu transposon into target DNA is available from Finnzymes Oy and is based upon the above-noted considerations. In the commercial Mu system, MuA transposase and the target DNA are mixed together ex vivo to form products that have a polynucleotide inserted into target DNA. The precut transposon ends of non-transferred strands are provided with four extra bases. Both transposon ends are right ends that include R1 and R2, with the final TT of R2 absent from the construct. The sequence of the right ends used in the commercial product, presented as SEQ ID NO:4, is:
gatcTGAAGCGGCGCACGAAAAACGCGAAAGCGTTTCACGATAAATGCGAAAAC 3′-ACTTCGCCGCGTGCTTTTTGCGCTTTGCCAAAGTGCTATTTACGCTTTTG-5′
The transposition products are subsequently delivered (e.g., by electroporation) into competent target cells. Cells that contain such extra-chromosomal transposition products are then selected using well-known methods. Transposition is completed outside of the target cell. In the commercial embodiment, the Mu transposon does not transpose into the DNA of the target cell.
Shoji-Tanaka, A., et al.,
B.B.R.C.
203:1756-1764 (1994) describe using purified retroviral integrase to mediate gene transfer into murine cells. Kuspa, A. and W. F. Loomis,
P.N.A.S. U.S.A.
89:8803-8807 (1992) and others have described specifically integrating a plasmid linearized with a restriction enzyme into a genomic restriction site by electroporating enzyme-cut nucleic acid along with the cleaving enzyme into target cells.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that a method for efficiently inserting a transposable polynucleotide at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a transposase protein complexed with (b) a polynucleotide that comprises (1) a pair of nucleotide sequences adapted for operably interacting with the transposase and (2) a transposable polynucleotide therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes, which are poised for transposition, from actually undergoing productive transposition. Synaptic complexes are then delivered into a target cell. In the target cell, the transposable polynucleotide is transposed into cellular target nucleic acid to form productive transposition products. Cells containing productive transposition products can be identified using a selection method such as antibiotic resistance.
The frequency of productive transposition of the transposable polynucleotide into the target nucleic acid can be enhanced by using in the method either a hyperactive transposase or a polynucleotide that comprises nucleotide sequences particularly well adapted for efficient transposition in the presence of the transposase, or both.
The present invention is further summarized in that a method for forming a library of cells that comprise insertional mutations includes the steps of combining in a plurality of target cells the cellular nucleic acid with the synaptic complex as described, and screening for cells that comprise insertional mutations.
In another aspect, the invention is further summarized as a library of cells that comprise insertional mutations formed according to the above-mentioned method. Such populations of cells that comprise random (or quasi-random) and independent mutational insertions in their genomes can be screened to select those cells that comprise an insertional mutation that induces a phenotypic or genotypic change relative to cells that were not subject to insertional mutagenesis.
It is an advantage of the present invention that the transposable polynucleotides used to form synaptic complexes can consist of transposon DNA apart from any flanking donor backbone (DBB) sequences. This is advantageous in that it reduces the likelihood of intramolecular transposition and increases the likelihood of transposition into a target genome. Moreover, eliminating DBB sequences from the polynucleotide simplifies preparation of the transposon sequences that can be used in the method.
It is another advantage of the present invention that the synaptic complex can form under conditions that disfavor non-productive intramolecular transposition events. This is advantageous in that substantially all of the synaptic complexes can undergo transposition when combined with the cellular DNA. Little, if any, of the nucleic acid in the synaptic complexes is inactive.
It is a feature of the present invention that transposition-promoting conditions are encountered only after the synaptic complex is in the presence of the target nucleic acid in the target cell.
Other objects, advantages, and features of the present invention will become apparent upon consideration of the following
Goryshin Igor Y.
Reznikoff William S.
Horlick Kenneth R.
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
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