WO2005049795A2 - Mammalian cell lines for detecting, monitoring, and optimizing oligonucleotide-mediated chromosomal sequence alteration - Google Patents

Abstract

The invention provides mammalian cell lines, kits comprising such cell lines, and methods of use for detecting, monitoring, and optimizing oligonucleotide-directed targeted chromosomal sequence alterations.

Description

MAMMALIAN CELL LINES FOR DETECTING, MONITORING, AND OPTIMIZING OLIGONUCLEOTIDE-MEDIATED CHROMOSOMAL SEQUENCE ALTERATION

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. provisional application no. 60/520,229, filed November 13, 2003, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] A number of methods have been developed to alter specific nucleotides within both isolated DNA molecules and DNA present within intact cells of bacteria, plants, fungi and animals, including humans. [0003] In one approach, genomic sequences are targeted for alteration by homologous recombination using duplex fragments. The duplex fragments are large, having several hundred basepairs . See, e.g., Kunzelmann et al . , Gene Ther. 3:859-867 (1996). [0004] In another approach, oligonucleotides are used to effect targeted genetic changes. [0005] In early experiments, oligonucleotide- directed sequence changes were typically effected in yeast, Moerschell et al . , 1988, Proc. Natl. Acad. Sci. 85:524 and Yamamoto et al., Yeast 8:935 (1992), which among eukaryotes are known to have high recombinogenic activity, although one series of experiments were attempted in human cells, Campbell et al . , The New Biologist 1: 223-227 (1989) .

[0006] More recently, a number of different types of polynucleotides and oligonucleotides have been described that permit targeted alteration of genetic material in cells of higher eukaryotes, including (i) triplex-forming oligonucleotides; (ii) chimeric RNA-DNA oligonucleotides that are ±nternally duplexed, notably in the region containing the nucleotide that directs the sequence alteration; and (iii) terminally modified single-stranded oligonucleotides having an internally unduplexed DNA domain and modified ends . [0007] Sequence-altering triple:xing oligonucleotides are described, for example, in U.S. Pat. Nos. 6,303,376, 5,962,426, and 5, 776, 744. [0008] Triplex-forming oligonucieotides require a structural domain that binds to a DNA helical duplex through Hoogsteen interactions between the major groove of the DNA duplex and the oligonucleotide . The binding domain must typically target polypurine or polypyri idine tracts. These sequence requirements limit the usefulness of triplex-forming oligonucleotides for targeted sequence alteration, requiring that the target sequence to be modified be situated in proximity to such polypurine or polypyrimidine tract. Triplex-forming oligonucleotides may also require an additional DNA reactive moiety, such as psoralen, to be covalently linked to the oligonucleotide, in order to stabilize the interactions between the triplex-forming domain of the oligonucleotide and the DNA double helix if the Hoogsteen interactions from the oligonucleotide/target base composition are insufficient. See, e.g., U.S. Patent 5,422,251. Such DNA-reactive moieties can, however, be indiscriminately mutagenic. [0009] In more recent work with sequence-altering triplexing oligonucleotides, the triplex-forming domain is linked or tethered to a domain that effects targeted alteration, Culver et al . , Nat. Biotechnology 17: 989- 93 (1999), relaxing somewhat the permissible distance between target sequence and polypurine/polypyrimidine stretch. [0010] Internally duplexed, hairpin- and double- hairpin-containing chimeric RNA-DNA oligonucleotides are described, inter alia, in U.S. Pat. Nos. 6,573,046; 5,888,983; 5,871,984; 5,795,972; 5,780,296; 5,760,012; 5,756,325; 5,731,181, and 5,565,350. Such chimeric RNA-DNA oligonucleotides are reportedly capable of directing targeted alteration of single base pairs, as well as introducing frameshift alterations, in cells and cell-free extracts from a variety of host organisms, including bacteria, fungi, plants and animals. The oligonucleotides are reportedly able to operate on almost any target sequence. [0011] Such chimeric molecules have significant structural requirements, however, including a requirement for both ribonucleotides and deoxyribonucleotides, and typically also a requirement that the oligonucleotide adopt a double-hairpin conformation. Even when such double hairpins are not - q -

required, however, significant structural constraints remain.

[0012] Single-stranded oligonucleotides having modified ends and an internally undupLexed DNA domain that directs sequence alteration are described in copending international patent applications published as WO 03/027265; WO 02/10364; WO 01/92512; WO 01/87914; and WO 01/73002, as well as in U.S. Pat. Nos. 6,479,292 and 6,271,360, the disclosures of which are incorporated herein by reference in their entireties. [0013] These single-stranded oligonucleotides have fewer structural requirements than chimeric oligonucleotides and are capable of directing sequence alteration, including introduction of fra eshift mutations, in cells and cell-free extracts from a variety of host organisms, including bacteria, fungi, plants and animals, in episomal and in chromosomal targets, often at alteration efficiencies that exceed those observed with hairpin-containing- , internally duplexed, chimeric oligonucleotides.

[0014] The usefulness of oligonucleotide-mediated nucleic acid sequence alteration — as a means, for example, for manipulating cloned DNA, for generating agricultural products with enhanced traits, for generating cellular models for laboratory use, or for generating animal models or animals with desired traits — is affected by its frequency and by the consistency with which it can be effected. The usefulness of oligonucleotide-mediated nucleic acid sequence alteration as an ex vivo or in vivo therapeutic method also depends on its efficiency and consistency. [0015] A continuing difficulty in efforts to achieve consistent, high efficiency, sequence alteration with any of the above-described sequence-altering oligonucleotides, however, particularly as applied to chromosomal sequence alteration in mammalian cells, has been the absence of a model system and assay method that rapidly, efficiently, and consistently provide and report sequence alteration events.

[0016] There thus exists a need in the art for a mammalian cellular model system and assay method that rapidly, efficiently, and consistently provide and report chromosomal sequence alteration events.

SUMMARY OF THE INVENTION [0017] The present invention solves these and other problems in the art by providing, in a first aspect, a mammalian cell line for detecting oligonucleotide- directed chromosomal sequence alteration events. [0018] The cell line comprises at least one chromosomally integrated copy of at least a first recombinant expression construct. The integrated construct is capable of expressing a first encoded marker protein. As integrated, however, the at least first construct has at least one mutation that renders the first encoded marker protein phenotypically undetectable or phenotypically distinguishable from wild type. Upon successful targeted sequence alteration, the marker protein as expressed provides the wild type marker phenotype. [0019] In particularly useful embodiments, the cell line is a human cell line, notably a human tumor cell line, such as DLD-1 or HTC-15, and the marker is a - fa-

protein having a GFP-like chromophore, such as enhanced GFP ("eGFP") .

[0020] In a second aspect, the invention provides a method of monitoring oligonucleotide-directed chromosomal sequence alteration events in a mammalian cell .

[0021] The method comprises detecting at least a first marker protein in a mammalian cell line of the present invention. The cell line has at least one chromosomally integrated copy of at least a first recombinant expression construct, the construct capable of expressing a first marker protein. Prior to oligonucleotide-directed chromosomal sequence alteration, the chromosomally integrated construct has at least one mutation that renders the first encoded marker protein phenotypically undetectable or phenotypically distinguishable from the wild type marker protein. [0022] In certain embodiments, the at least first marker protein is detected in a population of cells that are commonly contacted with a sequence altering oligonucleotide. The cells of the population can be separately detected, or detected together in a pool. In either set of embodiments, the detection method can usefully report a group statistic on marker abundance in the population, such as an average, a median, or a mode .

[0023] In some embodiments, in which the at least first marker protein has a GFP-like chromophore or a tetracysteine tag, the first marker protein is detected by fluorescence, such as by flow cytometry. [0024] In a further aspect, the invention provides a kit.

[0025] The kit comprises an aliquot of at least one cell line of the present invention, the cells capable of further propagation, and often of further replication.

[0026] In some embodiments, for example, the kit can include an aliquot of a DLD-1 cell line having an chromosomally integrated marker protein with a GFP-like chromophore, such as eGFP.

[0027] In a yet further aspect, the invention provides a method of oligonucleotide-directed alteration of the sequence of a predetermined chromosomal locus within a population of mammalian cells.

[0028] The method comprises concurrently targeting for oligonucleotide sequence alteration both a predetermined chromosomal locus and at least a first chromosomally integrated expression construct, wherein the construct is capable of expressing a first encoded marker protein. The at least first construct has at least one mutation that renders the first encoded marker protein phenotypically undetectable or distinguishable from wild type prior to oligonucleotide-directed chromosomal sequence alteration.

[0029] The next step comprises selecting a subpopulation of cells that detectably express the marker protein; and then identifying within the subpopulation of selected cells those having altered sequence at the predetermined locus. The selection can be by flow cytometry. [0030] The cell lines and kits of the present invention can further be used to optimize conditions for oligonucleotide-directed sequence alteration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which like characters refer to like parts throughout, and in which:

[0032] FIGS. 1A - IE show integration of DLD-1 cells with the eGFP vector, with FIG. 1A schematizing the structure of the integrative cassette and showing both mutant [SEQ ID NO: 1] and wild-type [SEQ ID NO: 2] eGFP sequences; FIG. IB showing sequencing data from sequencing of the wild type eGFP gene (TAC, on the left) and the mutant TAG (stop codon) eGFP gene (on the right) at the mutation site; FIG. 1C showing the sequence of sequence altering targeting oligonucleotides EGFP3S/72NT and EGFP3S/72T; FIG. ID showing fluorescence images of selected integrant clones, obtained with multiphoton confocal microscopy; and FIG. IE showing histograms generated from flow cytometric analysis for cell clone 4 (left) and clone 3 (right) integrated with wild type pEGFP-N3, according to the present invention; [0033] FIG. 2 presents a protocol for sequence alteration ("gene repair") in engineered DLD-1 cells, according to the present invention; [0034] FIG. 3. presents fluorescence images of sequence-altered cells obtained by confocal microscopy 2 days and 8 days respectively after gene alteration according to the present invention;

[0035] FIG. 4 presents fluorescence activated cell, sorting data demonstrating differences in sequence alteration efficiency in different eGFP mutant DLD clones, according to the present invention; and

[0036] FIG. 5 presents data demonstrating the strand bias of oligonucleotide-directed sequence alteration,, with FIG. 5A showing data obtained in DLD-1 clone 1 and FIG. 5B showing data obtained in DLD-1 clone 4, according to the present invention.

DETAILED DESCRIPTION

[0037] In a first aspect, the invention provides a mammalian cell line useful for detecting oligonucleotide-directed chromosomal sequence alteration events.

[0038] The cell line comprises at least one chromosomally integrated copy of at least a first recombinant expression construct, the construct being" capable of expressing a first encoded marker protein. As integrated, the construct has at least one mutation that renders the first encoded marker protein phenotypically undetectable or phenotypically distinguishable from the wild type marker protein.

[0039] The cell line can be from the cells of any mammal, and can thus be drawn from mammals used as laboratory model systems, such as rodents, including mice, rats, guinea pigs, lagomorphs such as rabbits, monkeys, apes, dogs, pigs, and cats; from livestock, such as cattle, bison, horses, goats, sheep, pigs, -ιu-

chickens, geese, ducks, turkeys, pheasant, ostrich and pigeon; or from other primates, including humans. [0040] Usefully, the cell line is an immortalized or fully transformed cell line, such as a neoplastic cell line .

[0041] For example, the cell line can be a human tumor cell line selected from the group consisting of lines commercially available from the American Type Culture Collection (ATCC, Manassas, VA, USA) (listed by ATCC reference number) :

CRL- ^■2327 CRL- -7850 CCL- -249 CRL-5972 CRL-7207 CRL- -7213 CRL- -7435 HTB- -78 CRL-7928 HTB-40 HTB- -111 CRL- -7911 HTB- -45 HTB-49 CRL-5892 HTB- -52 CRL- -5944 CRL- -5844 CRL-5866 CRL-5868 CRL- -5918 CRL- -5942 CRL- -7380 CRL-5877 CRL-5907 HTB- -57 CRL- -5850 CRL- -5852 CRL-5865 CRL-5872 CRL- -5876 CRL- -5881 CRL- -5882 CRL-5887 CRL-5897 CRL- -5909 CRL- -5911 CRL- -5912 CRL-5922 CRL-5936 CRL- -5895 CRL- -5985 HTB- -179 HTB-55 CRL-5889 CRL- ^■2351 CRL- -7222 CRL- -7226 CRL-7227 CRL-7245 CRL- -7253 CRL- -7477 CRL- -7480 CRL-7647 HTB-132 HTB- -27 HTB- -30 HTB- -128 HTB-130 HTB-21 HTB- -22 HTB- -26 HTB- -151 HTB-147 CRL-10303 HTB- ^■161 HTB- -75 HTB- -76 HTB-77 CRL-1687 CRL- -1997 CRL- -2119 CRL- -2172 HTB-79 HTB-80 CRL- -1682 CRL- -1435 CRL- -2422 CCL-235 CRL-1622 HTB- ^■112 HTB- -113 CRL- -7758 CRL-5883 CRL-5885 CRL- -5894 CRL- -2220 CRL- -5800 CRL-5810 CRL-5870 CRL- -5875 CRL- -5884 CRL- -5891 CRL-5896 CRL-5899 CRL- -5908 CRL- -5914 CRL- -5921 CRL-5935 CRL-5939 CRL- -5941 CRL- -5834 HTB- -178 CRL-10296 CRL-2170 CRL- -1579 CRL- -1585 HTB- -137 CRL-9267 HTB-56 CRL- -2461 CRL- -1718 HTB- -12 HTB-13 CRL-7762 CRL- -7646 CRL- -7649 CRL- -7672 CRL-2081 CRL-7588 CRL- -2294 CRL- -5807 CRL- -5835 CRL-7194 HTB-168 CRL- -1647 HTB- -62 CCL- -85 CCL-86 CCL-87 CRL- -1432 CRL- -1484 CRL- -1648 CRL-2392 CRL-2393 CRL- -2394 HTB- -61 CRL- -7933 CRL-7936 CRL-10237 CCL- -213 CCL- -214 HTB- -60 CRL-1596 CRL-1923 CRL- -2128 CRL- -1594 CRL- -1595 CRL-7396 CRL-7914 CRL- -7920 HTB- -31 HTB- -32 CRL-7908 HTB-44 CCL- -185 HTB- -53 CRL- -5867 CRL-7316 CRL-7336 CRL- -7365 CRL- -7368 CRL- -7721 CRL-1420 HTB-134 CCL- -138 CRL- -10995 CRL- -1740 CRL-2505 HTB-81 CRL- -5833 CRL- -7870 CRL- -1803 CRL-1472 CRL-1473 CRL- -7150 CRL- -7193 CRL- -7833 CRL-7926 CRL-7927 HTB- -9 CRL- -1671 HTB- -118 CCL-257 CRL-5804 CRL- -5808 CRL- -5817 CRL- -5821 CRL-5825 CRL-5828 CRL- -5832 CRL- -5836 CRL- -5837 CRL-5840 CRL-5841 CRL- -5842 CRL- -5846 CRL- -5849 CRL-5854 CRL-5855 CRL- -5856 CRL- -5861 CRL- -5862 CRL-5869 CRL-5871 CRL- -5886 CRL- -5888 CRL- -5898 CRL-5902 CRL-5906 CRL- -5910 CRL- -5916 CRL- -5920 CRL-5979 CRL-5824 CRL- -2049 CRL- -2062 CRL- -2064 CRL-2066 CRL-2177 CRL- -2195 CRL- -5853 CRL- -5858 CRL-5859 CRL-5864 CRL- -5874 CRL- -5879 CRL- -5901 CRL-5903 CRL-5905 CRL- -5913 CRL- -5927 CRL- -5929 CRL-5931 CRL-5932 CRL- -5933 CRL- -5934 CRL- -5940 CRL-5976 CRL-5978 CRL- -5982 CRL- -5983 CRL- -5984 HTB-119 HTB-120 HTB- -171 HTB- -172 HTB- -173 HTB-175 HTB-180 CRL- -5813 HTB- -184 CRL- -11351 CRL-5809 CRL-5811 CRL- -5823 CRL- -5831 CRL- -5845 HTB-177 HTB-183 CCL- -256 CRL- -5803 CRL- -5816 CRL-5818 CRL-7891 HTB- -94 CCL- -98 CRL- -7394 HTB-144 HTB-36 CRL- -1978 HTB- -46 HTB- -47 CCL-251 CCL-252 CCL- -253 CCL- -254 CCL- -218 CCL-220 CCL-220. CCL- -221 CCL- -222 CCL- -224 CCL-225 CCL-227 CCL- -228 CCL- -229 CCL- -230 CCL-231 CCL-233 CCL- -237 CCL- -238 CCL- -255 CL-187 CL-188 CRL- -2102 CRL- -7214 CRL- -7351 CRL-7352 HTB-37 HTB- -38 HTB- -39 CCL- -234 CRL-7159 CRL-7184 CRL- -7168 CCL- -250 CCL- -250.1 CRL-2134 CRL-2158 CRL- -2159 CCL- -247 CCL- -248 CRL-7399 CRL-7456 CRL- -7273 CRL- -2105 CRL- -8294 CRL-7252 CRL-7692 CRL- -7043 CRL- -7233 HTB- -186 CRL-2260 CRL-2558 CRL- -1918 CRL- -12420 CRL- -1500 CRL-1504 CRL-1897 CRL- -2320 CRL- -2338 CRL- -7345 HTB-121 HTB-129 HTB- -133 HTB- -20 HTB- -25 CRL-1837 HTB-104 CRL- -7802 CRL- -1550 CRL- -7932 HTB-33 HTB-34 CRL- -1555 CRL- -7902 CCL- -199 HTB-54 CRL-7228 CRL- -7905 CRL- ^■2592 HTB- -41 CRL-1469 CRL-2138 CRL- -7556 CRL- -7598 HTB- -166 CRL-7744 CRL-7951 CRL- -7062 CRL- -7287 CRL- -7508 CRL-7509 CRL-7664 CRL- -7665 CRL- -7824 HTB- -152 CRL-7666 CRL-7668 CCL- -121 CRL- -7604 HTB- ^■91 TIB-223 CRL-7773 CRL- -1739 CRL- -1863 CRL- ^■1864 CRL-5822 HTB-135 CRL- -5971 CRL- ^•5973 CRL- ^■5974 HTB-103 CRL-7447 CRL- -7473 CRL- ^•7554 CRL- ^■7579 CRL-7617 CRL-7081 -1-. -

CRL- -7547 CRL- -1620 CRL- -2020 CRL- -7899 HTB- -16

CRL- -1690 HTB- -14 HTB- -15 HTB- -138 CRL- -10741

CRL- -11997 CRL- -2233 CRL- -2234 CRL- -2235 CRL- -2236

HB-8064 HB-8065 CRL- -8024 CRL- -1532 CRL- -1533

CRL- -2367 CRL- -7593 CCL- -113 CRL- -7264 CRL- -7362

CRL- -7488 HTB- -146 CRL- -7779 CRL- -7373 CRL- -7378

CRL- -2175 CCL- -244 CRL- -7428 CRL- -7630 CRL- -7629

CRL- -5878 CRL- -2262 CRL- -2289 CRL- -5923 CRL- -7822

HTB- -88 CRL- -10423 HTB- -92 CRL- -7306 CRL- -7313

CRL- -7818 CRL- -7218 CRL- -2230 CRL- -2231 CRL- -2277

CRL- -8119 HTB- -142 CRL- -11622 CRL- -12043 HTB- -176

TIB- -161 CRL- -7235 CRL- -7507 CRL- -11213 CRL- -8543

CRL- -7797 TIB- -162 CRL- -7755 CRL- -7641 HTB- -105

HTB- -106 CRL- -2365 CRL- -2366 CRL- -7684 CRL- -7685

HTB- -64 HTB- -63 CRL- -1424 CRL- -1619 CRL- -1872

CRL- -1974 CRL- -7691 HTB- -65 HTB- -66 HTB- -67

HTB- -68 HTB- -69 HTB- -70 HTB- -71 HTB- -72

HTB- -73 CRL- -2407 CRL- -11732 CRL- -1973 CRL- -8805

HTB- -185 HTB- -187 CRL- -7724 CRL- -7426 CRL- -7568

CRL- -11147 CRL- -1675 CRL- -1676 CRL- -2500 CRL- -7299

CRL- -7360 CRL- -7425 CRL- -7572 CRL- -7585 CRL- -7637

CRL- -7653 CRL- -7654 CRL- -7658 CRL- -7686 CRL- -7687

CRL- -7690 CRL- -7898 CRL- -7904 CRL- -9446 CRL- -9451

CRL- -9607 HTB- -140 HTB- -114 HTB- -115 CRL- -5820

CRL- -5915 CRL- -5917 CRL- -5938 CRL- -1848 CRL- -8644

CCL- -127 CRL- -2137 CRL- -2142 CRL- -2149 CRL- -2266

CRL- -2267 CRL- -2268 CRL- -2270 CRL- -2271 CRL- -2273

HTB- -11 CRL- -5893 HTB- -10 HTB- -148 CRL- -7434

CRL- -10236 CRL- -2261 CRL- -2408 CRL- -2073 CRL- -7609

CRL- -11226 CRL- -1423 CRL- -1427 CRL- -1543 CRL- -1544

CRL- -1545 CRL- -1546 CRL- -1547 CRL- -7005 CRL- -7023

CRL- -7060 CRL- -7134 CRL- -7140 CRL- -7263 CRL- -7444

CRL- -7448 CRL- -7471 CRL- -7489 CRL- -7511 CRL- -7521

CRL- -7537 CRL- -7543 CRL- -7577 CRL- -7595 CRL- -7600

CRL- -7602 CRL- -7606 CRL- -7622 CRL- -7626 CRL- -7628

CRL- -7631 CRL- -7632 CRL- -7633 CRL- -7634 CRL- -7642

CRL- -7643 CRL- -7644 CRL- -7645 CRL- -7765 CRL- -7766

CRL- -7780 CRL- -7783 CRL- -7823 CRL- -7939 CRL- -7943

CRL- -8303 CRL- -8304 HTB- -85 HTB- -96 CRL- -2098

CRL- -7677 CRL- -5819 CRL- -7573 CCL- -155 CRL- -8033-1

CRL- -8083 CRL- -8147 TIB- -196 CRL- -9068 CRL- -2237

CRL- -2238 CRL- -5904 CRL- -7753 CRL- -2335 CRL- -1902

CRL- -2314 CRL- -2315 CRL- -2316 CRL- -2321 CRL- -2322

CRL- -2324 CRL- -2326 CRL- -2329 CRL- -2331 CRL- -2336

CRL- -2340 CRL- -2343 CRL- -11730 CRL- -11731 CRL- -2380

CRL- -2330 CCL- -105 CRL- -1231 CRL- -1611 CRL- -1932

CRL- -1933 CRL- -1440 CRL- -7678 CRL- -7239 HTB- -169 HTB--18 CRL--7713 CRL--7726 CRL--7763 CRL--7767 CRL- -7774 CRL- -7862 CCL- -136 CRL- -1598 CRL- -2061 CRL- -7752 CRL- -7900 HTB- -153 HTB- -82 CRL- -7910 CRL- -7732 CRL- -7746 HTB- -86 CRL- -7037 CRL- -7482 CRL- -7800 CRL- -7030 CRL- -7085 CRL- -10302 HTB- -35 CRL- -5928 HTB- -182 HTB- -59 HTB- -58 CCL- -30 HTB- -43 HTB- -107 CRL- -1623 CRL- -1624 CRL- -1628 CRL- -1629 CRL- -2095 HTB- -3 HTB- -117 CRL- -5826 CRL- -7289 CRL- -7440 HTB- -93 CRL- -1572 CRL- -7886 CRL- -7882 CRL- -1749 CRL- -2169 HTB- -1 HTB- -4 HTB- -5 HTB- -2 CRL- -1649 CRL- -1976 CRL- -2274 CRL- -1977 CRL- -7102 CRL- -1593. 2 CRL- -8033-2

[0042] For example, the cell line can be a human carcinoma cell line, such as a human adenocarcinoma cell line, including a colorectal adenocarcinoma cell line, such as DLD-1 (ATCC CCL-221) or HCT-15 (ATCC CCL- 225) . [0043] At least one copy of the first expression construct is integrated into the chromosomes of the cell line. In some embodiments, 2, 3, 4 or even 5 or more copies are integrated. In other embodiments, as many as 6, 7, 8, 9, 10 or more copies are integrated. [0044] Integration is typically performed at random sites in the cellular genome, although in other embodiments integration can be targeted via homologous recombination. [0045] In certain embodiments, a second recombinant expression construct is integrated into the cell line chromosomes .

[0046] The second construct in these embodiments is capable of expressing a second encoded marker protein . The second construct can ^' optionally have at least one mutation that renders the second encoded marker protein phenotypically undetectable or distinguishable from wild-type marker protein.

[0047] In embodiments in which a second (or optional third or more) expression construct is integrated into the chromosomes of the cell line, the second (and optional third or more) marker proteins are usefully phenotypically distinguishable from the first marker protein.

[0048] For example, in embodiments in which the first and second (and optional further additional) marker proteins are fluorescent, or can be rendered fluorescent, the markers are usefully fluorescently distinguishable. Such distinction can be in excitation spectra, emission spectra, or both. [0049] The first (and optional second or further) construct-encoded marker protein can usefully be protein having a GFP-like chromophore or a tetracysteine tag. [0050] As used herein, "GFP-like chromophore" refers to an intrinsically fluorescent protein moiety comprising an 11-stranded β-barrel (β-can) with a central α-helix, the central α-helix having a conjugated π-resonance system that includes two aromatic ring systems and the bridge between them. By "intrinsically fluorescent" is meant that the GFP-like chromophore is entirely encoded by its amino acid sequence and can fluoresce without requirement for cofactor or substrate. [0051] The GFP-like chromophore can be selected from GFP-like chromophores found in naturally occurring proteins, such as A. ictoria GFP (GenBank accession number AAA27721) , Renilla reniformis GFP, FP583 (GenBank accession no. AF168419) (DsRed) , FP593 (AF272711), FP483 (AF168420) , FP484 (AF168424), FP595 (AF246709), FP486 (AF168421) , FP538 (AF168423) , and FP506 (AF168422), and need include only so much of the native protein as is needed to retain the chromophore ' s intrinsic fluorescence. Methods for determining the minimal domain required for fluorescence are known in the art. Li et al . , "Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence," J. Biol. Chem. 272:28545-28549 (1997) .

[0052] Alternatively, the GFP-like chromophore can be selected from GFP-like chromophores modified from those found in nature. Typically, such modifications are made to improve recombinant production in heterologous expression systems (with or without change in protein sequence) , to alter the excitation and/or emission spectra of the native protein, to facilitate purification, to facilitate or as a consequence of cloning, or are a fortuitous consequence of research investigation .

[0053] The methods for engineering such modified GFP-like chromophores and testing them for fluorescence activity, both alone and as part of protein fusions, are well-known in the art. Early results of these efforts are reviewed in Heim et al., Curr . Biol. 6:178- 182 (1996), incorporated herein by reference in its entirety; a more recent review, with tabulation of useful mutations, is found in Palm et al . , "Spectral Variants of Green Fluorescent Protein, " in Green

Fluorescent Proteins, Conn (ed.), Methods Enzymol . vol. 302, pp. 378 - 394 (1999), incorporated herein by reference in its entirety.

[0054] A variety of such modified chromophores are now commercially available and can readily be used in the fusion proteins of the present invention.

[0055] For example, EGFP ("enhanced GFP"), Cormack et al., Gene 173:33-38 (1996); U.S. Pat. Nos. 6,090,919 and 5,804,387, is a red-shifted, human codon-optimized variant of GFP that has been engineered for brighter fluorescence, higher expression in mammalian cells, and for an excitation spectrum optimized for use in flow cytometers . EGFP can usefully contribute a GFP-like chromophore to the fusion proteins of the present invention. A variety of EGFP vectors, both plasmid and viral, are available commercially (Clontech Labs, Palo Alto, CA, USA) , including vectors for bacterial expression, vectors for N-terminal protein fusion expression, vectors for expression of C-terminal protein fusions, and for bicistronic expression. [0056] Toward the other end of the emission spectrum, EBFP ("enhanced blue fluorescent protein") and BFP2 contain four amino acid substitutions that shift the emission from green to blue, enhance the brightness of fluorescence and improve solubility of the protein, Heim et al . , Curr. Biol. 6:178-182 (1996); Cormack et al . , Gene 173:33-38 (1996). EBFP is optimized for expression in mammalian cells whereas BFP2, which retains the original jellyfish codons, can be expressed in bacteria; as is further discussed below, the host cell of production does not affect the utility of the resulting fusion protein. The GFP-like chromophores from EBFP and BFP2 can usefully be included in the fusion proteins of the present invention, and vectors containing these blue-shifted variants are available from Clontech Labs (Palo Alto, CA, USA) . [0057] Analogously, EYFP ("enhanced yellow fluorescent protein") , also available from Clontech Labs, contains four amino acid substitutions, different from EBFP, Ormo et al., Science 273:1392-1395 (1996), that shift the emission from green to yellowish-green. Citrine, an improved yellow fluorescent protein mutant, is described in Heikal et al . , Proc. Natl. Acad. Sci. USA 97:11996-12001 (2000). ECFP ("enhanced cyan fluorescent protein") (Clontech Labs, Palo Alto, CA, USA) contains six amino acid substitutions, one of which shifts the emission spectrum from green to cyan. Heim et al . , Curr . Biol. 6:178-182 (1996); Miyawaki et al., Nature 388:882-887 (1997). The GFP-like chromophore of each of these GFP variants can usefully be included in fusion protein aggregants of the present invention.

[0058] The GFP-like chromophore can also be drawn from other modified GFPs, including those described in U.S. Pat. Nos. 6,124,128; 6,096,865; 6,090,919; 6,066,476; 6,054,321; 6,027,881; 5,968,750; 5,874,304; 5,804,387; 5,777,079; 5,741,668; and 5, 625, 048, the disclosures of which are incorporated herein by reference in their entireties.

[0059] See also Proc. Natl. Acad. Sci USA 97:11984; J. Biol. Chem. 276:29621; Nat. Biotechnol. 20:83; J. Biol. Chem. 277:7633; Proc. Natl. Acad. Sci USA

99:7877; Biotechniques 33:592; Bioconjug. Chem. 11:65; Biochem. J. 355:1-12; Martynov et al . , "A purple-blue - l b -

chromoprotein from goniopora tenuidens belongs to the

DsRed subfamily of GFP-like proteins," J. Biol. Chem.

2003 Sep 15 [Epub ahead of print]; Biophys . J.

85(3):1839, the disclosures of which are incorporated herein by reference in their entireties.

[0060] For example, the protein marker can be eGFP.

[0061] In other embodiments, the marker protein can have a tetracysteine tag.

[0062] Recently, a new class of fluorophore derivatives of tremendous value in the detection, visualization, and purification of recombinant proteins has been introduced.

[0063] These biarsenical fluorophore derivatives, as adducts with EDT (1, 2-ethanedithiol) , are self- quenching: the small size of the EDT moieties permits free rotation of the arsenic atoms, which quenches fluorescence of the fluorophore. The essentially nonfluorescent molecule is rendered fluorescent by competitive displacement of the EDT moiety by a specific tetracysteine peptide motif (CCXXCC, where "X" represents any amino acid except cysteine) , an engineered sequence that is uncommon in natural proteins; binding to the tetracysteine motif constrains motion of the arsenic atoms, unquenching the fluorophore. Griffin et al . , Science 281:269 (1998);

Griffin et al . , Methods Enzymol . 327:565-78 (2000);

Adams et al . , J. Amer . Chem . Soc . 124:6063-6076 (2002);

Gaietta et al . , Science 503-507 (2002); U.S. Pat. Nos.

5,932,474, 6,054,271; 6,451,569; 6,008378; U.S. patent application publication no. 2003/0083373, and international patent application publication no. WO 99/21013, the disclosures of which are incorporated herein by reference in their entireties . [0064] Advantages of the biarsenical fluorophores as fluorescent protein labeling reagents include small size, ability of the EDT₂ adducts to cross cell membranes, ability to recognize a binding domain that is sufficiently small as not to interfere substantially with the function of the protein to which it is fused, nanomolar (or lower) dissociation constant for binding to the tetracysteine motif, rapid conversion from a nonfluorescent to a fluorescent state upon binding, and the reversibility of its binding upon addition of a high concentration (millimolar) of EDT. [0065] The biarsenical derivative of fluorescein that is most commonly used is 4 ' -5 ' -bis (1, 3, 2- dithioarsolan-2-yl) fluorescein- (2, 2-ethanedithiol) ₂ , known as FlAsH -EDT₂ or Lumio Green, and is available commercially (Invitrogen Corp., Carlsbad, CA) . The red-fluorescing biarsenical resorufin derivative, known as ReAsH or Lumio Red, is also available commercially; methods of synthesizing other such biarsenical fluorophores, such as CHoXAsH-EDT₂ and HoXAsH-EDT₂ are described in the literature. [0066] Tetracysteine biarsenical affinity tags (FlAsH tags) have been successfully incorporated at either the N- or C-termini of proteins, as well as exposed surface regions within a protein and have been used to permit visualization of recombinant proteins expressed within living cells, and in SDS-PAGE gels. Griffin et al . 1998, Griffin et al . 2000, Adams et al . 2002, supra . -Z Ό~

[0067] In embodiments in which a plurality of expression constructs, each encoding a separate marker protein, are chromosomally integrated, the marker proteins usefully can be proteins having GFP-like chromophores that are spectrally distinguishable from one another.

[0068] The mutation in the chromosomally integrated recombinant construct can provide a null phenotype -- that is, render the encoded marker phenotypically undetectable -- or can render the mutant marker protein distinguishable from the wild type protein. [0069] The mutation can be a single point mutation, either in the coding region or promoter, or can be a plurality of mutations. [0070] For example, at least one mutation can contribute to a premature stop codon within the coding region, leading to expression of a truncated marker protein that is phenotypically undetectable. [0071] In some embodiments, the wild type marker protein is not only detectable, but phenotypically selectable or screenable. Usefully, the marker protein is phenotypically selectable or screenable within living cells. For example, in presently preferred embodiments, the wild type marker protein is flow cytometrically selectable or screenable.

[0072] In a second aspect, the invention provides a method of monitoring oligonucleotide-directed chromosomal sequence alteration events in a mammalian cell. The method comprises detecting at least a first marker protein in an engineered mammalian cell line.

The cell line has at least one chromosomally integrated copy of at least a first recombinant expression construct, the construct being capable of expressing a first marker protein.

[0073] As integrated into the cell line chromosome, the integrated construct has at least one mutation that renders the first encoded marker protein phenotypically undetectable or distinguishable from wild-type. Successfully targeted sequence alteration events are thus detectable by phenotypic appearance of the marker protein, or by appearance of the distinguishable wild type marker phenotype against a background of mutant phenotype. Detection can be quantitative or qualitative .

[0074] In typical embodiments, the cell line is as described above. [0075] The sequence-altering oligonucleotide can be any oligonucleotide that is capable of altering chromosomal nucleic acid sequences, including (i) triplex-forming oligonucleotides; (ii) chimeric RNA-DNA oligonucleotides that are internally duplexed, notably in the region containing the nucleotide that directs the sequence alteration; and (iii) terminally modified single-stranded oligonucleotides having an internally unduplexed DNA domain and modified ends. [0076] Terminally-modified sequence altering single-stranded oligonucleotides with internally unduplexed domains ("single stranded targeting oligonucleotides") are typically at least about 17 nucleotides in length, and often no more than about 121 nucleotides in length, with intermediate lengths, such as 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides permissible. [0077] The internally unduplexed domain is typically at least 8 contiguous deoxyribonucleotides . [0078] These single stranded targeting oligonucleotides are fully complementary in sequence to the sequence of a first strand of the nucleic acid target, but for one or more mismatches as between the sequences of the internally unduplexed deoxyribonucleotide domain and its complement on the target nucleic acid first strand. Typically, each of the mismatches is positioned at least 8 nucleotides from each of the oligonucleotide' s 5' and 3' termini. [0079] The single stranded targeting oligonucleotides have at least one terminal modification. In certain embodiments, the modification is usefully selected from phosphorothioate internucleoside linkage, 2'-0-alkyl nucleotide, such as a 2'-0-methyl nucleotide, or locked nucleic acid (LNA) . [0080] The oligonucleotide can include a plurality of such modifications at one or both termini. In some embodiments, the oligonucleotide has three phosphorothioate linkages at each of its termini. [0081] In one series of embodiments, the marker protein is detected in a population of cells that are commonly contacted with at least one sequence altering oligonucleotide . [0082] In various embodiments, the detection method reports a group statistic on wild type marker abundance in the population, whether or not the cells are individually queried. In such cases, the group statistic can conveniently be an average, a median, or a mode .

[0083] In some of these embodiments, each of the cells in the population is separately detected. [0084] For example, the marker protein can have a GFP-like chromophore or a tetracysteine tag and the fluorescence detected in individual cells of the population by flow cytometry.

[0085] In a further aspect, the invention provides a kit for detecting oligonucleotide-directed chromosomal sequence alteration events in a mammalian cell.

[0086] The kit comprises an aliquot of a first cell line according to the present invention. The cells are provided in a form in which they are capable of further propagation; often, in a form in which they are capable of further replication.

[0087] In one embodiment, the cell line is a DLD-1 cell line with a recombinant expression construct encoding a protein with mutant GFP-like chromophore, such as a truncated fluorescent protein, such as eGFP. [0088] The kit can further comprise an oligonucleotide species, optionally in liquid composition, that is capable of effecting targeted correction of the mutation in the marker construct. [0089] The kit can optionally further comprise a second cell line that has at least one chromosomally integrated copy of at least a second recombinant expression construct. -..4 -

[0090] In embodiments in which the second expression construct is capable of expressing a wild type marker protein, the cells can be used as a positive detection control, such as a flow cytometry positive control. In other embodiments, the second expression construct includes a mutation that creates a null phenotype or a marker protein that is phenotypically distinguishable from wild type. [0091] The kits can further comprise instructions for performing the methods of the present invention. [0092] The cell lines of the present invention, whether alone or as a component of a kit, provide a consistent, high efficiency, rapid system for monitoring the efficacy of oligonucleotide-directed targeted sequence alteration.

[0093] The cell lines and kits thus have a variety of uses.

[0094] For example, in a further aspect, the present invention provides a method of oligonucleotide-directed alteration of the sequence of a predetermined chromosomal locus within a population of mammalian cells .

[0095] As further described in copending U.S. patent application no. 10/681,074, filed filed October 7, 2003, the disclosure of which is incorporated herein by reference in its entirety, the frequency of oligonucleotide-directed sequence alterations at a first nucleic acid target site is higher in a population of cells that has been selected for concurrent alteration at a second nucleic acid target site, as compared to a population of cells that has not been selected for concurrent alteration at a second nucleic acid target site.

[0096] Thus, in one series of embodiments, the method of this aspect of the present invention can usefully comprise concurrently targeting for oligonucleotide sequence alteration both a predetermined chromosomal locus and at least a first chromosomally integrated expression construct in a mammalian cell line; the construct is capable of expressing a first encoded marker protein, the at least first construct having at least one mutation that renders the first encoded marker protein phenotypically undetectable or distinguishable from wild type prior to oligonucleotide-directed chromosomal sequence alteration.

[0097] In a second step, a subpopulation of cells are selected that detectably express the wild type marker protein, and then cells within this subpopulation are identified that have altered sequence at the predetermined locus.

[0098] In embodiments in which the marker protein is fluorescent or can be made fluorescent — such as a protein having a GFP-like chromophore or having a tetracysteine tag — selection can be performed using flow cytometry, such as fluorescence activated cell sorter .

[0099] The cell lines and kits of the present invention can also be used to optimize conditions for oligonucleotide-directed targeted sequence alteration. [0100] For example, the characteristics of the sequence-altering oligonucleotide — e . g. its length, the position within the oligonucleotide of the -2 b-

correcting mismatch or mismatches, the chemical composition, such as terminal modifications; the transfection conditions, such as electroporation conditions; the effect of suppression of or enhanced expression of various cellular proteins (such as those described in copending U.S. patent application nos. 10/351,662, filed January 24, 2003 and 10/260,375, filed September 27, 2002, the disclosures of which are incorporated herein by reference in their entireties) ; the effect of treating the cells with various chemical compounds (such as those described in copending U.S. patent application no. 10/384,918, filed March 7, 2003, the disclosure of which is incorporated herein by reference in its entirety, including hydroxyurea and histone deacetylase inhibitors such as trichostatin A) ; and other treatments and factors that affect the efficiency of targeted sequence alteration, can now readily be assessed by determining the conversion efficiency in the cell lines of the present invention under such varying conditions.

[0101] As an example, using the cell lines of the present invention, different DNA transfer vehicles, such as PEI proteins, liposomes, and electroporation, can be compared and optimized. [0102] As a further example, further described in

Examples 1 and 2 below, simply changing the density of the cells in the electroporation chamber can influence the repair frequency drastically, and the cell lines of the present invention can be used to optimize such conditions.

[0103] As a yet further example, Examples 1 and 2 further demonstrate that the cell lines of the present invention can be used to assess the contribution of target strand selection, demonstrating a strand bias effect when the high density (100 μl volume) electroporation conditions are used, with the data indicating that the nontranscribed strand is more amenable to targeting than the transcribed strand (see FIGS. 5A and 5B) .

[0104] In yet another example, the cell lines of the present invention can be used to assess the effect on recombination efficiency of mutations in cellular proteins that mediate the sequence alteration event, such as RAD51, as further described in U.S. application no. 10/861,178, filed June 4, 2004, incorporated herein by reference in its entirety.

[0105] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1 Materials and Methods

Cell line and culture conditions

[0106] The DLD-1 cell line is obtained from ATCC (American Type Culture Collection, Manassas, VA) and grown in RPMI 1640 medium with 2 mM L-glutamine, 4.5g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and supplemented with 10% fetal bovine serum (FBS) .

Construction of DLD-1 cells with integrated eGFP gene [0107] pEGFP-N3 plasmid is purchased from Clontech (Palo Alto, CA) . pEGFP plasmid carries an enhanced green fluorescent protein (eGFP) gene and a neo gene, -Z ϋ -

so the transformant clone integrating this eGFP gene can be selected with G418 (GIBCO, Invitrogen Corporation, Carlsbad, CA.).

[0108] A single base substitution is introduced into the eGFP gene harbored in the pEGFP-N3 to create a premature stop codon (eGFP Y291X) , which inactivates green fluorescent protein. Briefly, a pair of primers carrying the desired point mutation in eGFP are annealed to the sequence of the eGFP gene to make a point mutation at position 875 of pEGFP-N3 with the

QuikChange site-directed mutagenesis Kit (Stratagene, La Jolla, CA) . The primers used in this experiment are: forward primer: 5 ' -GACCTACGGCGTGCAGTGC-3 ' [SEQ ID NO.: 3]; backward primer: 5 ' -ACGCCGTAGGTCAGGGTG G-3* [SEQ ID NO: 4] .

[0109] An aliquot of 10⁶ cells is resuspended in 0.8 ml of serum free RPMI 1640 media and electroporated with 5 μg of pEGFP-N3 by ECM®830 instrument (BTX, a division of Genetronics, Inc., San Diego, CA) . [0110] Cells are grown in 10 ml of fresh complete media with 300μg/ml G418 in 100 mm Petri dishes. After 14 days of culture with G418 selection, G418-resistant single colonies are transferred to 24-well plates for additional 3 days with G418 selection following 3 days without G418 selection. Finally, the stable transformant clones carrying neo gene are maintained at 200μg/ml of G418.

[0111] Integration of eGFP is confirmed by PCR and the copy number of eGFP gene determined by Southern blotting. eGFP sequence-al tering oligonucleotides [0112] FIG. 1C shows the sequence of targeting oligonucleotides EGFP3T/72NT [SEQ ID NO.: 5] and EGFP3T/72T [SEQ ID NO.: 6]. [0113] EGFP3S/72NT and EGFP3S/72T are single- stranded oligonucleotides, 72 nucleotides long, with "NT" representing the oligonucleotide that targets (is complementary to) the non-transcribed strand of the mutant eGFP gene and "T" representing the oligonucleotide that targets (is complementary to) the transcribed strand. Asterisks indicate phosphorothioate linkages within the oligonucleotides, located at both 3' and 5' termini. The two oligonucleotides are synthesized with three terminal phosphorothioate linkages at each terminus.

[0114] A sequence-nonspecific terminally modified 74 nt oligonucleotide (Hyg3S/74NT; SEQ ID NO: 7), bearing no complementarity to the mutant eGFP target, is illustrated below the specific ones.

eGFP gene targeting

[0115] Cells grown in complete medium supplemented with 10% FBS are trypsinized and harvested by centrifugation at 1500 rpm for 5 minutes. The cell pellet is resuspended in fresh serum-free medium at a density of 2^χl0⁶ cells/lOOμl.

[0116] A lOOμl aliquot of cells is mixed with 20μg of 72 base single stranded oligonucleotide containing three phosphorothioate linkages at both the 3' and 5' termini, and are transferred into a 4 mm gap cuvette (Fisher Scientific; Pittsburgh PA) followed by electroporation under the following conditions: voltage = 250V pulse length = 13 msec pulse = 2 interval=l second unipolar .

[0117] Cells are incubated on ice for 10 minutes and then seeded onto 60 mm Petri dish containing 3 ml fresh media supplemented with 10% FBS . Afterward, cells are kept in an incubator for 40 hours before proceeding to flow cytometric analysis.

Flow cytometric analysis [0118] The cells will emit fluorescence if the eGFP mutant gene in the chromosome of DLD-1 is converted by oligonucleotide-directed targeted sequence alteration into wild type. Flow cytometry is used to detect the converted cells. [0119] Cells are washed with PBS once and collected by trypsinization and centrifugation, followed by resuspension in 1 ml FACS buffer (0.5% BSA, 2 mM EDTA, pH 8.0, 2μg/ml propidium iodide). Cells are incubated at room temperature for 30 min. Converted cells are measured by Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, Rutherford, NJ) . The frequency of converted cells was calculated by CellQuest and GFP/PI software programs. EXAMPLE 2 Results

Construction of cell lines [0120] DLD-1 are epithelial cells which originated from a colorectal adenocarcinoma, exhibiting a normal colon epithelial cell cycle/doubling time of 19 hours but of unknown passage. The mutation in the eGFP gene is located at residue 67, converting a tyrosine codon to a TAG (stop) codon.

[0121] FIG. 1A shows the structure of the integrated expression construct. Wild type and mutant sequences of the eGFP gene are shown in FIG. IB (left and right panels, respectively) . [0122] Essentially as described in Example 1, five μg of pEGFP-N3 (wild type) is electroporated into 10⁶ cells for integration and the cells then transferred to 100 mm Petri dish containing 10 ml fresh complete media with 300μg/ml G418. After 14-days of culture with G418 selection, G418 resistant single colonies are transferred to 24-well plates to enrich under G418 selection; the transformant clones are maintained at 200μg/ml of G418. [0123] Fluorescence images are then obtained with multiphoton confocal microscopy.

[0124] As shown in FIG. ID, the lines differ dramatically in eGFP expression as assessed by confocal microscopy.

FACs as an assay for eGFP gene repair [[00112255]] AApppprrooxxiimmaatteellyy llxxllOO⁵⁵ cceellllss aaire washed with PBS and collected by trypsinization and centrifugation - Z.-

Cells are stained in 1 ml of PBS-FACS buffer for 30 minutes and 50,000 cells counted by Becton Dickinson FACSCalibur flow cytometer. Events represent the number of cells separated by the flow cytometer. [0126] FIG. IE shows histograms (dot plots) from flow cytometric analysis for wild type cell clone 4 (left panel) and wild type clone 3 (right panel) integrated with wild type pEGFP-N3, with propidium iodide fluorescence on the Y axis and eGFP fluorescence on the X axis. The dot plots are divided into four quadrants, as follows.

[0127] LR (low right quadrant) : the number of live cells with eGFP expression; LL (low left quadrant) : the number of live cells without eGFP expression; UR (upper right quadrant) : the number of dead cells with eGFP expression; UL (upper left quadrant) : the number of dead cells without eGFP expression.

[0128] Notwithstanding the dramatic difference in fluorescence detected between wild type clones 4 and 3 by confocal microscopy (see FIG. ID, upper right and lower right panels), the results obtained by flow cytometry are much more consistent as between these clones (compare left and right panels of FIG. IE) . Flow cytometry, which is capable of individually querying cells for fluorescence emission, and is also able to provide group statistics, thus is superior in consistency to earlier assays using microscopic examination: flow cytometry analysis provides a moderating effect on the assay variation as approximately 80% of each cell line was found to express eGFP; levels of eGFP are often detectable by FACS, but not by confocal visualization. Sequence alteration

[0129] Clones containing mutant eGFP are created and expanded into the test cell lines used for our experiments .

[0130] We have chosen to focus on two lines, clone 1 and clone 4, because of their robust growth and their capacity to survive electroporation. Southern blot analyses reveal that clone 1 harbors 2-4 copies of the fusion construct while clone 4 contains a single copy of the target gene (data not shown) .

[0131] Prior to examining gene repair activity, we establish the timing of eGFP expression and find that integrated DLD-1 clonal lines maximally express wild- type eGFP 40-48 hours after plating (data not shown) . [0132] FIG. 2 presents the protocol for targeted sequence alteration of a mutant eGFP gene integrated in the DLD-1 cell line. [0133] Based on experiments with the wild type eGFP integrants, the protocol in FIG. 2 includes FACS analysis 48 hours after addition of sequence altering oligonucleotide by electroporation. [0134] We also measure cell viability for each experiment using FACS profiles in propidium iodide. The percentage of converted cells in a whole population is then calculated by CellQuest (Becton Dickinson) and GFP/PI programs.

[0135] For all experiments, 2 x 10⁶ cells are electroporated with sequence altering or control oligonucleotide and 10⁵ cells prepared for FACS; dual analyses for correction and viability enables all cells undergoing electroporation to be followed with positive cells (eGFP⁺) identified without selection. [0136] Experiments are carried out using two mutant clonal cell lines - clone 1 and clone 4 - both of which have confirmed integrated targets. [0137] The oligonucleotide EGFP3S/72NT is electroporated into either clone and results of the correction are presented in Table 1 and FIG. 4.

[0138] Both clones display gene repair of the eGFP mutation, but at significantly different levels while the cell viability remains quite consistent as between the clones. Each clone is tested three times and the standard deviation reflects very reproducible results. [0139] The correction efficiency is determined by dividing the number of eGFP positive cells by the number of cells analyzed in each experiment (usually 50,000 cells). As a baseline control, we find that wild-type eGFP is generally expressed in greater than 75% of the cells (data not shown) , consistent with the data presented in Figure IE.

[0140] Our results show that clone 1 has the capacity to enable correction of the mutant eGFP at a 15-fold higher frequency than that enabled in the clone 4 cells.

[0141] FIG. 3 presents confocal images of the clone 1 (experiment number 2) cells 2 days and 8 days after electroporation and the results indicate that the correction of the eGFP mutation is inheritable.

Strand bias and the electroporation reaction [0142] We use two different oligonucleotides to direct repair in order to examine strand bias in correction.

[0143] The first oligonucleotide, EGFP3S/72NT, targets (is complementary to) the nontranscribed strand, while the second vector, EGFP3S/72T, targets (is complementary to) the transcribed strand. The oligonucleotides are introduced at increasing levels into clone 1 and the correction efficiencies measured 48 hours later.

[0144] Both oligonucleotides support gene repair (Table 2) but at significantly different levels.

Table 2

Electroporation carried out in an 800 μl volume [0145] The numbers in parentheses indicate the effective concentration of EGFP3S/72NT in the electroporation chamber. The results shown are the average of three independent experiments with standard deviation calculated by Microsoft Excel. [0146] The NT vectors outperform the T vectors at each dosage. [0147] Thus, targeting the nontranscribed strand produces a higher level of correction than targeting the transcribed strand. The same strand bias is also seen in experiments performed using clone 4; here the NT strand is favored 10-30-fold (data not shown) . [0148] The experimental protocol for gene repair also involves an electroporation step which we optimize in a 100 μl volume (2xl0⁶ cells) . Under these conditions, the cells are compacted enough to receive a maximal dosage of oligonucleotide while maintaining a high level of viability. [0149] As shown in Table 2, however, increasing the amount of oligonucleotide in the reaction does not elevate the correction efficiency. If, however, the volume in which the electroporation is carried out is expanded 8-fold (800 μl) , a dose response is observed (Table 2) indicating that a greater level of oligonucleotide is needed to direct maximum levels of correction when the cell density and/or the effective oligonucleotide concentration in the electroporation chamber is reduced.

[0150] When the effective concentrations are similar (5 μM from the 100 μl volume and 3.75 μM from the 800 μl volume) , a similar level of gene repair is observed. [0151] Such subtle variables among research labs clearly contribute to the lack of robust results with this technique.

[0152] We have chosen to utilize 20 μg in the 100 μl electroporation volume to keep the overall amount of oligonucleotide required in the experiment to a minimum, lowering the potential for toxic effects.

[0153] All patents and publications cited in this specification are herein incorporated by reference as if each had specifically and individually been incorporated by reference herein. Although the foregoing invention has been described in some detail by way of illustration and example, it will be readily apparent to those of ordinary skill in the art, in light of the teachings herein, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims .

Claims

WHAT IS CLAIMED IS:

1. A mammalian cell line for detecting oligonucleotide-directed chromosomal sequence alteration events, the cell line comprising: at least one chromosomally integrated copy of at least a first recombinant expression construct, said at least first construct capable of expressing a first encoded marker protein, said at least first construct having at least one mutation that renders the first encoded marker protein phenotypically undetectable or phenotypically distinguishable.

2. The cell line of claim 1, wherein said mammalian cell line is a human cell line.

3. The cell line of claim 2, wherein said human cell line is a neoplastic cell line.

4. The cell line of claim 3, wherein said human cell line is a carcinoma cell line.

5. The cell line of claim 4, wherein said carcinoma cell line is an adenocarcinoma cell line.

6. The cell line of claim 5, wherein said adenocarcinoma cell line is a colorectal adenocarcinoma cell line.

7. The cell line of claim 6, wherein said cell line is DLD-1 or HCT-15.

8. The cell line of claim 7, wherein said cell line is DLD-1.

9. The cell line of any one of claims 1 - 8, wherein at least two copies of said first recombinant expression construct are integrated into the chromosomes of said cell line.

10. The cell line of claim 9, wherein at least three copies of said first recombinant expression construct are integrated into the chromosomes of said cell line.

11. The cell line of any one of claims 1 - 10, further comprising at least one chromosomally integrated copy of at least a second recombinant expression construct, said at least second construct capable of expressing a second encoded marker protein, said at least second construct having at least one mutation that renders the second encoded marker protein phenotypically undetectable.

12. The cell line of claim 11, wherein the wild type of said first and second encoded protein markers are phenotypically distinguishable.

13. The cell line of claim 12, wherein the wild type of said first and second encoded protein markers are spectrally distinguishable.

14. The cell line of any one of claims 1 - 13, wherein said at least first construct-encoded marker protein is a protein having a GFP-like chromophore or a tetracysteine tag.

15. The cell line of claim 14, wherein said at least first protein is eGFP.

16. The cell line of any one of claims 1 - 14, wherein said at least one mutation contributes to a premature stop codon within the coding region of said at least first encoded marker protein.

17. The cell line of any one of claims 1 - 16, wherein the wild type of said at least first marker protein is phenotypically selectable or screenable.

18. The cell line of claim 17, wherein the wild type of said at least first marker protein is phenotypically selectable or screenable within a living cell .

19. The cell line of claim 18, wherein the wild type of said at least first marker protein is flow cytometrically selectable or screenable.

20. A method of monitoring oligonucleotide- directed chromosomal sequence alteration events in a mammalian cell, the method comprising: detecting at least a first marker protein in a mammalian cell line having at least one chromosomally integrated copy of at least a first recombinant expression construct, the construct capable of expressing a first marker protein, wherein said chromosomally integrated construct has at least one mutation that renders the first encoded marker protein phenotypically undetectable or distinguishable from wild type prior to oligonucleotide-directed chromosomal sequence alteration.

21. The method of claim 20, wherein said at least first marker protein is detected in a population of cells commonly contacted with a sequence altering oligonucleotide .

22. The method of claim 21, wherein each of the cells in said population is separately detected.

23. The method of claim 21 or claim 22, wherein said detecting reports a group statistic on marker abundance in said population.

24. The method of claim 23, wherein said group statistic is an average, a median, or a mode.

25. The method of any one of claims 20 - 24, wherein said cell line is a cell line of any one of claims 1 - 20.

26. The method of any one of claims 20 - 24, wherein said at least first marker protein has a GFP- like chromophore or a tetracysteine tag and said first marker protein is detected by fluorescence.

27. The method of claim 26, wherein said fluorescence detection is performed by flow cytometry.

28. A kit for detecting oligonucleotide- directed chromosomal sequence alteration events in a mammalian cell, the kit comprising: an aliquot of a first cell line according to any one of claims 1 - 19, the aliquot containing cells capable of further propagation.

29. The kit of claim 28, wherein said cell line is a DLD-1 cell line and said first marker protein has a GFP-like chromophore.

30. The kit of claim 29, wherein said GFP- like chromophore is eGFP.

31. The kit of any one of claims 28 - 30, wherein said cell line further comprises at least one chromosomally integrated copy of at least a second recombinant expression construct, said at least second construct capable of expressing a second encoded marker protein, said at least second construct having at least one mutation that renders the second encoded marker protein phenotypically undetectable.

32. The kit of claim 31, further comprising an oligonucleotide capable of altering the mutation in said second integrated construct to render said second encoded marker protein phenotypically detectable.

33. The kit of any one of claims 28 - 32, further comprising: a control cell line, said control cell line having at least one chromosomally integrated copy of at least a first recombinant expression construct, said at least first construct capable of expressing a first encoded marker protein.

34. The kit of claim 33, wherein said control cell line first marker protein is the wild type of said first cell line.

35. The kit of any one of claims 28 - 34, further comprising: instructions for performing the method of any one of claims 20 - 27.

36. A method of oligonucleotide-directed alteration of the sequence of a predetermined chromosomal locus within a population of mammalian cells, the method comprising: concurrently targeting for oligonucleotide sequence alteration both a predetermined chromosomal locus and at least a first chromosomally integrated expression construct, wherein said construct is capable of expressing a first encoded marker protein, said at least first construct having at least one mutation that renders the first encoded marker protein phenotypically undetectable prior to oligonucleotide-directed chromosomal sequence alteration; selecting for a subpopulation of cells that detectably express said marker protein; and then identifying within the subpopulation of selected cells those having altered sequence at said predetermined locus.

37. The method of claim 36, wherein said cells are the cells of any one of claims 1 - 19.

38. The method of claim 36 or 37, wherein said selecting is by flow cytometry.

39. The method of claim 38, wherein said marker is a protein having a GFP-like chromophore or a tetracysteine tag.

40. The method of claim 39, wherein said marker is eGFP.