Genetically Modified Cell Lines Including a TP53 Modification and Methods of Use

Abstract

The present disclosure is directed to genetically engineered cell lines which include a modification to knockout a portion of the TP53 gene. Embodiments disclosed herein provide aspects of the knockout cell lines, methods for producing the knockout cell lines, in vitro assays using the knockout cell lines, and kits including the knockout cell lines. In certain implementations, the embodiments can provide doctors and patients improved tools for determining a treatment or for comparing treatments for patients having tumors that include a TP53 mutation.

Claims

1. A knockout cell line, wherein each cell of the knockout cell line comprises a MCF7 breast cancer cell having a deletion of at least one coding region in a gene, and wherein the gene includes a TP53 gene having a nucleotide sequence corresponding to Seq. ID No. 1.

2. The knockout cell line of claim 1, wherein the coding region comprises a part of an exon.

3. The knockout cell line of claim 1, wherein the coding region comprises one or more of exons 1-11.

4. The knockout cell line of claim 3, wherein each of exons on 1-11 include an nucleotide sequence from seq. ID No. 1, and wherein the nucleotide sequence corresponds to a range of base numbers for each exon selected from the group: the nucleotide sequence for exon 1 comprises base numbers 1-162, the nucleotide sequence for exon 2 comprises base numbers 10917-11018, the nucleotide sequence for exon 3 comprises base numbers 11136-11157, the nucleotide sequence for exon 4 comprises base numbers 11267-11545, the nucleotide sequence for exon 5 comprises base numbers 12303-12486, the nucleotide sequence for exon 6 comprises base numbers 12568-12680, the nucleotide sequence for exon 7 comprises base numbers 13249-13358, the nucleotide sequence for exon 8 comprises base numbers 13702-13838. the nucleotide sequence for exon 9 comprises base numbers 13931-14004, the nucleotide sequence for exon 10 comprises base numbers 16824-16930, the nucleotide sequence for exon 11 comprises base numbers 17849-19137.

5. The knockout cell line of claim 4, wherein the MCF7 breast cancer cell includes a deletion of the nucleotide sequence for exon 4, and wherein the nucleotide sequence for exon 4 comprises: TABLE-US-00005 (SEQIDNO:5) TCCCCCTTGCCGTCCCAAGCAATG GATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGT TCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCC AGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCT ACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGT CATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTA CGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAG TCTGTGACTTGCACG

6. The knockout cell line of claim 4, wherein the MCF7 breast cancer cell includes a deletion of the nucleotide sequence for exon 10, and wherein the nucleotide sequence for exon 10 comprises: TABLE-US-00006 (SEQIDNO:6) ATCCGTGGGCGTGAGCG CTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTC AAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGG CTCACTCCAG

7. An in vitro assay for determining efficacy of a treatment in breast cancer cells that include a TP53 gene mutation, comprising: providing the treatment to a plurality of cells having a deletion of at least one coding region in the TP53 gene, and measuring a result.

8. The in vitro assay of claim 7, further comprising: providing the treatment to a plurality of wild type MCF7 breast cancer cells; measuring a wild type result; and comparing the wild type result to the result.

9. The in vitro assay of claim 8, wherein comparing the wild type result to the result comprises: determining a first quantitative measurement describing the number of live wild type MCF7 breast cancer cells included in the plurality of wild type MCF7 breast cancer cells to which the treatment was provided; determining a second quantitative measurement describing the number of live cells included in the plurality of cells having a deletion of at least one coding region in the TP53 gene to which the treatment was provided, and wherein the treatment provided to the wild type MCF7 breast cancer cells and the treatment provided to the plurality of cells having the deletion of at least one coding region in the TP53 gene are the same.

10. The in vitro assay of claim 7, wherein measuring the result comprises determining a quantitative measure of cell death.

11. The in vitro assay of claim 7, wherein providing the treatment comprises administering a drug to the plurality of cells derived from the knockout cell line.

12. The in vitro assay of claim 11, wherein the drug comprises one or more of the drugs from the group consisting of: Abiraterone, Afatanib, Alectinib, Allopurinol, Altretamine, Amifostine, Aminolevulinic Acid, Anastrozole, Arsenic Trioxide, Axitinib, Azactidine, Belinostat, Bendamustine Hydrochloride, Bleomycin Sulfate, Bortezomib, Bosutinib, Busulfan, Cabazitaxel, Cabozantinib, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Celecoxib, Ceritinib, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Clyclopamine, Cobimetinib, Crizotinib, Cyclophosphamide, Cytarabine Hydrochloride, Dabrafenib Mesylate, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Dexrazoxane, DMSO, Docetaxel, Doxorubicin Hydrochloride, Enzalutamide, Epirubicin Hydrochloride, Erismodgib, Erlotinib Hydrochloride, Estramustine Phosphate Sodium, Etoposide, Everolimus, Exemestane, Floxuridine, Fludarabine Phosphate, Fluorouracil, Fulvestrant, Gefintinib, Gemcitabine Hydrochloride, Hydroxyurea, Ibrutinib, Idarubicin Hydrochloride, Idelalisib, Ifosfamide, Imatinib, Imiquimod, Irinotecan Hydrochloride, Ixabepilone, Ixazomib, Laptinib, Lenalidomide, Letrozole, Lomustine, Mechlorethamine Hydrochloride, Megestrol Acetate, Melphalan Hydrochloride, Mercaptopurine, Methotrexate, Methoxsalen, Mitomycin, Mitotane, Mitoxantrone, Nelarabine, Nilotinib, Nutlin3, Olaparib, Omacetaxine Mepesuccinate, Osimertinib, Oxaliplatin, Paclitaxel, Palbociclib, Panobinostat, Pazopanib Hydrochloride, Pemetrexed Disodium Salt Heptahydrate, Pentostatin, Pipobroman, Plerixafor, Plicamycin, Pomalidomide, Ponatinib, Pralatrexate, Procarbazine Hydrochloride, Raloxifene, Regorafenib, Rom idepsin, SenexinB, Sirolimus, Sorafenib, Streptozocin, Sunitinib, Tamoxifen Citrate, Temozolomide, Temsirolimus, Teniposide, Thalidomide, Thioguanine, Thiotepa, Topotecan Hydrochloride, Trametinib, Tretinoin, Triethylenemelamine, Trifluridine, Uracil Mustard, Valrubicin, Vandetanib, Vemurafenib, Vinblastine Sulfate, Vincristine Sulfate, Vinorelbine Tartrate, Vismodegib, Vorinostat, Zoledronic Acid 2.

13. A method for producing a knockout cell line from a wild type cell line, the method comprising deleting a portion of the TP53 gene in a cell derived from the wild type cell line, wherein deleting the portion of the TP53 gene comprises delivering a guide RNA to the cell.

14. The method of claim 13, wherein the wild type cell line comprises human MCF7 breast cancer cells.

15. The method of claim 13, wherein the portion of the TP53 gene comprises at least one coding region included in exons 4-10.

16. The method of claim 13, wherein delivering the guide RNA to the cell includes delivering an expression cassette to the cell, wherein the expression cassette includes a DNA sequence for expressing the guide RNA.

17. The method of claim 16, wherein delivering the guide RNA to the cell further includes delivering a second expression cassette to the cell, wherein the second expression cassette includes a DNA sequence for expressing Cas9.

18. The method of claim 13, further comprising selecting for a genetically modified cell, wherein selecting for the genetically modified cell comprises culturing the cell in the presence of an agent.

19. The method of claim 18, wherein the agent comprises Nutlin3.

20. The method of claim 13, wherein the guide RNA comprises one or more of the sequences: TABLE-US-00007 (SEQIDNO:7) CCATTGTTCAATATCGTCCG, (SEQIDNO:8) GACGGAAACCGTAGCTGCCC, and (SEQIDNO:9) TGGTTATAGGATTCAACCGG.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0016] A full and enabling disclosure of the present invention, including the best mode thereof to one skilled in the art, is set forth more particularly in the remainder of the specification, which includes reference to the accompanying figures, in which:

[0017] FIGS. 1A and 1B illustrate example genetic modifications to the TP43 gene included in certain embodiments of the disclosure.

[0018] FIG. 2 illustrates a gel in accordance with embodiments of the disclosure.

[0019] FIG. 3 illustrates a sequence comparison in accordance with embodiments of the disclosure.

[0020] FIG. 4 illustrates a gel in accordance with an embodiment of the disclosure.

[0021] FIG. 5 illustrates a sequence comparison in accordance with an embodiment of the disclosure.

[0022] FIG. 6 illustrates a sequence comparison in accordance with an embodiment of the disclosure.

[0023] FIG. 7 illustrates a gel in accordance with an embodiment of the disclosure.

[0024] FIG. 8 illustrates a graph displaying relative cell number vs. log[conc] for example knockout cell lines in accordance with an embodiment of the disclosure.

[0025] FIG. 9 illustrates a graph displaying area under the curve (AUC) for TP53 knockout (KO) pools vs. AUS TP53 for wild type (WT.)

[0026] FIGS. 10A-10D illustrate graphs displaying relative cell number vs. log[conc] for example knockout cell lines in accordance with an embodiment of the disclosure.

[0027] FIGS. 11A-11C illustrate graphs displaying a drug resistance vs. nutlin resistance. The drugs are respectively: oxaliplatin, SFU, and Palb.

[0028] Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention.

DETAILED DESCRIPTION

[0029] Reference now will be made to embodiments of the invention, one or more examples of which are set forth below. Each example is provided by way of an explanation of the invention, not as a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as one embodiment can be used on another embodiment to yield still a further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied exemplary constructions.

[0030] The present disclosure is directed to genetically engineered cell lines which include a modification to knockout a portion of the TP53 gene. Embodiments disclosed herein provide aspects of the knockout cell line, methods for producing the knockout cell line, in vitro assays using the knockout cell line, and kits including the knockout cell line. In certain implementations, the embodiments can provide doctors and patients improved tools for determining a treatment or for comparing treatments for patients having tumors that include a TP53 mutation.

[0031] An example embodiment of the disclosure can include a knockout cell line composed of MCF7 breast cancer cells having decreased endogenous expression of at least one coding region in the TP53 gene. Generally, the cells of the knockout cell line include a genetic modification to remove or delete a portion of the TP53 gene which results in decreased endogenous expression.

[0032] In embodiments of the disclosure, the at least one coding region can include one or more of exons 4-10 in the TP53 gene. In some embodiments, the at least one coding region can include exon 4. In certain embodiments, the at least one coding region can include exon 4 and exon 5. In some embodiments, the at least one coding region can include all of exons 4-10.

[0033] For embodiments of the disclosure, the genetic modification can be applied to a native cell line (i.e., wild type). In an example implementation, the native cell line can include human MCF7 breast cancer cells, and an example embodiment can include a genetically modified MCF7 cell line having a genetic modification to remove or delete a portion of the TP53 gene. In another example implementation, the native cell line can include 600MPE, AU565, and/or BT-483. Generally, any cell line including a native TP53 gene can be genetically modified to produce a knockout cell line.

[0034] Several non-limiting examples of knockout cell lines disclosed herein include: an MCF7 cell line that includes a deletion of one or more of exons 4-10 of the TP53 gene; an AU565 cell line that includes a deletion of one or more of exons 4-5 of the TP53 gene; and a BT-483 cell line that includes a deletion of one or more exons 6-10 of the TP53 gene. These examples are provided for illustrative purposes to demonstrate how combinations of cell lines and genetic modifications may be produced using this disclosure.

[0035] Another example embodiment of the disclosure can include an in vitro assay for determining the efficacy of a treatment in cancer cells that include a TP53 gene mutation. In an example embodiment, the assay can include providing the treatment to a group of cells from a knockout cell line containing a TP53 gene mutation. In an implementation, the group of cells can be derived from any of the knockout cell lines disclosed herein (e.g., a MCF7 breast cancer cell having decreased endogenous expression of at least one coding region in the TP53 gene). During and/or after providing the treatment, the in vitro assay can further include measuring a result. In certain embodiments, measuring the result can include determining a quantitative measure of cell death (e.g., H&E staining). In some embodiments, the in vitro assay can also include providing the treatment to a group of cells from the native cell line (e.g., the MCF7 cell line). In these embodiments, the in vitro assay can also include comparing the treatment to the group of cells from the native cell line. As an example implementation, comparing the treatment to the cells from the native cell line can include determining a first quantitative measurement describing or approximating the number of live cells from the native cell line to which the treatment was provided, and determining a second quantitative measurement describing or approximating the number of live cells from the knockout cell line to which the treatment was provided. In some implementations, the first quantitative measurement and the second quantitative measurement can include a statistic, the statistic indicating if the first quantitative measurement is significantly different (e.g., higher or lower) compared to the second quantitative measurement.

[0036] For embodiments of the disclosure that include an in vitro assay, providing the treatment can include administering a drug to a group of cells from the knockout cell line. Generally, any drug can be used. Table 2 includes a list of example drugs; however, it should be understood that the list in Table 2 is not intended to be limiting and other drugs, both known and undiscovered, may be used in embodiments of the disclosure. Additionally, administering the drug can include administering one or more drugs, for example administering one or more of the drugs: Nutlin3, Fluorouracil, and Palbociclib.

[0037] A further embodiment can include a kit for assessing a treatment for a patient diagnosed with breast cancer. In an implementation, the kit can include an assay including a well-plate containing cells from a native cell line and knockout cells from a knockout cell line (the knockout cell line formed by deleting or inactivating a portion of the TP53 gene in the native cell line). In an example implementation, the cells from a native cell line can include MCF7 breast cancer cells and knockout cells can include cells derived from a knockout MCF7 cell line (including a genetic modification to one or more of exons 4-10 of the TP53 gene.) The kit can further include an indicator for measuring cell viability. In an example embodiment, the indicator can display a change in appearance (e.g., producing a color) when in contact with dead cells. Additionally, the change in appearance may be quantitative such that the intensity of the change in appearance can be related to the number of dead cells. The kit can also include the treatment (e.g., one or more drugs). In an implementation, the treatment can include Nutlin3, Fluorouracil, and Palbociclib

[0038] An additional embodiment of the disclosure includes a method for producing a knockout cell line from a native cell line, the method including deleting a portion of the TP53 gene in the native cell line. Generally, deleting a portion of the TP53 gene includes delivering a guide RNA to the native cell line and selecting for cells including the genetic modification. Example native cell lines may include: MCF7, 600MPE, AU565, and/or BT-483. Additionally, the portion of the TP53 gene can include at least one of exons 4-10.

[0039] In some implementations, delivering the guide RNA to the cell can include delivering an expression cassette to the cell, the expression cassette including a DNA sequence for expressing the guide RNA. In certain implementations, delivering the guide RNA to the cell can also include delivering a second expression cassette, including a DNA sequence expression Cas9. Several example guide RNAs for targeting the TP53 gene can include the sequences: CATTGTTCAATATCGTCCG, GACGGAAACCGTAGCTGCCC, and TGGTTATAGGATTCAACCGG.

[0040] To determine cells that have incorporated the genetic modification, a selection can be performed in some embodiments. In an example implementation, selecting for the genetically modified cell can include culturing the cells to which the guide RNA has been delivered in the presence of an agent. Exemplary agents can include any drugs to which cells derived from the native cell line are more sensitive compared to knockout cells, including a genetic modification to the TP53 gene. For example, a method for producing a TP53 knockout from the MCF7 cell line can include delivering a guide RNA to a group of cells derived from the native cell line and selecting for genetically modified cells by culturing the group of cells in the presence of Nutlin3.

[0041] Embodiments of the disclosure and examples described herein may be better understood with reference to the Sequence Listing filed with this disclosure. The Sequence Listing includes Seq ID No. 1, providing a nucleotide sequence for the TP53 gene for a Homo sapiens. Information regarding the sequence may be found from the NCBI database using the gene ID: ENSG00000141510 and transcript ID: ENST00000269305. The sequence listing also includes Seq ID No. 2 which provides an example knockout genetic sequence as observed in KO 5.6 examples. The sequence listing also includes Seq. ID No. 3 which provides an example knockout genetic sequence as observed in KO 3.4 examples.

Example 1

[0042] Example 1 discusses various methods and provides exemplary embodiments that may be understood in conjunction with the Drawings and Description provided herein. The materials and conditions described in the example are demonstrative and are not meant to constrain the scope of the disclosure only to the materials and conditions used.

Materials and Methods

Cell Lines Culture

[0043] Human MCF7 breast adenocarcinoma cells (ATCC HTB-22) and their derivatives were maintained at 37 C., 5% CO2 in DMEM (Gibco, Cat. No. 11995-065) with 100 ug/mL penicillin & 100 ug/mL streptomycin (Sigma, Cat. No. P4333), 10% FBS, 50 mM Sodium pyruvate (Sigma, Cat. No. S8636), 1% GlutaMAX (ThermoFisher, Cat. No. 35050061), and 10 ug/mL insulin. MCF7 cells were passaged every 4 to 7 days to maintain sub-confluence. All cell lines were maintained in culture for a maximum of 30 passages.

[0044] Genetic Sequence TP53

[0045] The genetic sequence used for the TP53 gene is provided below using 1 letter base convention to represent the individual nucleotides (i.e., adenosine, A; guanosine, G; Cytidine, C; and thymidine, T.) The first nucleotide, base number 1, is G and the subsequent nucleotides increment by 1 base number thereon. The number appearing on the left margin represents the base number of the first nucleotide on the line. Additionally, the sequence is from a Homo sapiens (human.)

TABLE-US-00001 Seq.IDNo.1: 1 GTTTTCCCCTCCCATGTGCTCAAGACTGGCGCTAAAAGTTTTGAGCTTCTCAAAAGTCTA 61 GAGCCACCGTCCAGGGAGCAGGTAGCTGCTGGGCTCCGGGGACACTTTGCGTTCGGGCTG 121 GGAGCGTGCTTTCCACGACGGTGACACGCTTCCCTGGATTGGGTAAGCTCCTGACTGAAC 181 TTGATGAGTCCTCTCTGAGTCACGGGCTCTCGGCTCCGTGTATTTTCAGCTCGGGAAAAT 241 CGCTGGGGCTGGGGGTGGGGCAGTGGGGACTTAGCGAGTTTGGGGGTGAGTGGGATGGAA 301 GCTTGGCTAGAGGGATCATCATAGGAGTTGCATTGTTGGGAGACCTGGGTGTAGATGATG 361 GGGATGTTAGGACCATCCGAACTCAAAGTTGAACGCCTAGGCAGAGGAGTGGAGCTTTGG 421 GGAACCTTGAGCCGGCCTAAAGCGTACTTCTTTGCACATCCACCCGGTGCTGGGCGTAGG 481 GAATCCCTGAAATAAAAGATGCACAAAGCATTGAGGTCTGAGACTTTTGGATCTCGAAAC 541 ATTGAGAACTCATAGCTGTATATTTTAGAGCCCATGGCATCCTAGTGAAAACTGGGGCTC 601 CATTCCGAAATGATCATTTGGGGGTGATCCGGGGAGCCCAAGCTGCTAAGGTCCCACAAC 661 TTCCGGACCTTTGTCCTTCCTGGAGCGATCTTTCCAGGCAGCCCCCGGCTCCGCTAGATG 721 GAGAAAATCCAATTGAAGGCTGTCAGTCGTGGAAGTGAGAAGTGCTAAACCAGGGGTTTG 781 CCCGCCAGGCCGAGGAGGACCGTCGCAATCTGAGAGGCCCGGCAGCCCTGTTATTGTTTG 841 GCTCCACATTTACATTTCTGCCTCTTGCAGCAGCATTTCCGGTTTCTTTTTGCCGGAGCA 901 GCTCACTATTCACCCGATGAGAGGGGAGGAGAGAGAGAGAAAATGTCCTTTAGGCCGGTT 961 CCTCTTACTTGGCAGAGGGAGGCTGCTATTCTCCGCCTGCATTTCTTTTTCTGGATTACT 1021 TAGTTATGGCCTTTGCAAAGGCAGGGGTATTTGTTTTGATGCAAACCTCAATCCCTCCCC 1081 TTCTTTGAATGGTGTGCCCCACCCCGCGGGTCGCCTGCAACCTAGGCGGACGCTACCATG 1141 GCGTGAGACAGGGAGGGAAAGAAGTGTGCAGAAGGCAAGCCCGGAGGTATTTTCAAGAAT 1201 GAGTATATCTCATCTTCCCGGAGGAAAAAAAAAAAGAATGGGTACGTCTGAGAATCAAAT 1261 TTTGAAAGAGTGCAATGATGGGTCGTTTGATAATTTGTCGGAAAAACAATCTACCTGTTA 1321 TCTAGCTTTGGGCTAGGCCATTCCAGTTCCAGACGCAGGCTGAACGTCGTGAAGCGGAAG 1381 GGGCGGGCCCGCAGGCGTCCGTGTGGTCCTCCGTGCAGCCCTCCGGCCCGAGCCGGTTCT 1441 TCCTGGTAGGAGGCGGAACTCGAATTCATTTCTCCCGCTGCCCCATCTCTTAGCTCGCGG 1501 TTGTTTCATTCCGCAGTTTCTTCCCATGCACCTGCCGCGTACCGGCCACTTTGTGCCGTA 1561 CTTACGTCATCTTTTTCCTAAATCGAGGTGGCATTTACACACAGCGCCAGTGCACACAGC 1621 AAGTGCACAGGAAGATGAGTTTTGGCCCCTAACCGCTCCGTGATGCCTACCAAGTCACAG 1681 ACCCTTTTCATCGTCCCAGAAACGTTTCATCACGTCTCTTCCCAGTCGATTCCCGACCCC 1741 ACCTTTATTTTGATCTCCATAACCATTTTGCCTGTTGGAGAACTTCATATAGAATGGAAT 1801 CAGGCTGGGCGCTGTGGCTCACGCCTGCACTTTGGGAGGCCGAGGCGGGCGGATTACTTG 1861 AGGATAGGAGTTCCAGACCAGCGTGGCCAACGTGGTGAATCCCCGTCTCTACTAAAAAAT 1921 ACAAAAATTAGCTGGGCGTGGTGGGTGCCTGTAATCCCAGCTATTCGGGAGGGTGAGGCA 1981 GGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCAAGATCGTGCCACTACAC 2041 TCCAGCCTGGGCGACAAGAACGAAACTCCGTCTCAAAAAAAAGGGGGGAATCATACATTA 2101 TGTGCTCATTTTTGTCGGGCTTCTGTCCTTCAATGTACTGTCTGACATTCGTTCATGTTG 2161 TATATATCAGTATTTTGCTCCTTTTCATTTAGTATAGTCCATCGATTGTATATCCGTCCT 2221 TTTGATGGCCTTTTGAGTTGTTTCCCATTTGCGGTTATGAAATAAAGCTGCTATAAACAT 2281 TCTTGTACAATTCTTTTTGTGATCATATGTTTTCGTGTTTCTTGGAGAAATACTTAGGAG 2341 GGGAATTGCGAGTTTGGAAGTAAAAAGTAGCTGTATTTTGAACTTTTTCAGAAGCTCTGA 2401 GTTTTCCAGAGCGGTTGTACCATTTTACACTCCAACTAGCAAGGTATGGGAGTTATTATG 2461 GTTGTGCCACAGCCTTCCGGACATTAGGTATTGTCAGTCTTTCTAATGTGGTATATCCTT 2521 GTGGTTGTAATTTACAGTTCTCTATTGACTAAGGATGTTCAGCATTTTTTCATGTGCCTA 2581 TTGGCCATTCGTATTTTGTTTGTAAAGTAGCTCTTCGAGTCTTTTACCTGTTATTTTGGT 2641 TTTTTGTTTGTTTTTATTGTTCAGTTGTGGGACTGCTTTATACATTCTGGATACAAGTCC 2701 TTTATCAGATCCATGTGTCGTGAATGTTTTCTTCTGATCTGTTGCTTGCCTATTTGTTTG 2761 CTTTACAGAGTTTACAGTATCTTAAGAGGAGTGGATTTATCTTTTTTATGTTCAGTATTT 2821 GCCTTGTCCTGTTTAGGACATCTTTTTTTTTTTTTTTAACCCCAGGGTCATGAAGATATT 2881 ATCTTACATTTTCTTTTAGGACCTTTATGGTTGTAAGTTTTACAGTAAGGTCCTTGAGCC 2941 ATTAATTAATTCTTAAAATTAATTGTTTATGGTGTGAGGTGTAGGAGTCAGTCTCTGGTA 3001 TCTTTCCTGTATGGAAATCCAGTTATTCTGTCTCCACTTGTTGAAATAGGCTTCCTTTCT 3061 CTACTGAATGCTTTTAATTTTAATTATTTTACAGTTGGAGTATAGGGCTACCATTTTAGT 3121 GCTATTTTCTTTTTTTCTTTGTTAATTTTTGAGACAGGGACTCACACTGTTGCCCAGGCT 3181 AGAGTACAATGGCACAATCAAGGCTTACTGCAGCCTCGAACCCCTGGGCTCAAGCAGTCC 3241 TCTAGCAGCCTCACGAGTAGCTGGGATTACTCCACCACACCCAGCTAACTATTTTATTTT 3301 TTTGTATTGACAGGATCTCACTATGTTGCCCAGGCTGGTCTCAAACTGCTGGCCTCAAGC 3361 TTTCATCCCATCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCATGCCTGA 3421 CCTCTTAGTGCTATTTTCTATTTATCTCCTCTGTTCTCTGCTCTCTTTAAACGTTGGAGG 3481 AAGAAACAGTACCCATCTTACACAAACTCTTCAGAAAACAGAGGAACAGACTGGGCGCGG 3541 TGGCTCATACCTGTAATCTCAGCACTTTGGTACGCTGAGGCAGGGGATCATTTGAGGTCG 3601 GGAGTTCGAGACCAGCCTGGCCAACACGGCGAAACCCCATCTCTACTAAAAATACAAAAA 3661 GTAGCTAGGCGTGGTGACACATACCTGTAATGCCAGTTACTCAGGAGGCTGAGGCACAAG 3721 AATCCCTTGAACCTGGGAAGCGGAGGTTGCAGTGAGCCGAGATTGCGCCACTGCACTCCA 3781 GCCTGGGCAACAGAGTGAGACCCTGTCTCAGAAAAAAAAAGAAAGAAAGAAAAAATAGAG 3841 GAATATTTCCCAACTTGTTTTCGAAGCCAGCATAATCCTGGTACCAAAACCAAACAAGGA 3901 CATTATAAGAAAAGAAAATATAGACCAATATTCCTGTTAGCATAGACATGCAACAGCTAA 3961 CCAATTTTAGCAAACCAAACCTGGTAATATAGAAAAAAGGATAAATAGGCCAGTCGCGGT 4021 GGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGCAGATCACTTGAGGTCA 4081 GGAGTTTGAGACCAGCCTGACCAACATGGTGAAACCCCGTTTCTAATAAAAATACAAAAA 4141 TCAGGCTGGGCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGC 4201 AGATCACGAGGTCAGGAGTTCAAGACCAGCCTGACCAATGTGGTGAAACGCCATCTCTAC 4261 TAAAAATACAAAAATCAGCCGGTGTGGTGGCACCTGCCTGTAATCCCAGCTACTCAGGAG 4321 GCTGAGGCAGAATTGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCAAGATCGTGCCA 4381 CTGCACTCCAGCCTGGGCGACAGAGCAAGACTTCATCTCAAAAAAAAAAAAAAATTAGCT 4441 GGGCATGGTGGTGGGCACCTGAAATCCCAGCTACTCGGGAGTCTGAGGCAGGAGAATCGC 4501 TTGAACCCAGGAGGCAGAAGTTGCACTGAGCTGGGATCACACCATTGCACTCCAGCCTGG 4561 GCAACAGAGTGAGACTCCATCTCAAAAAAAGAAAAAGAAAAAGGATAAATACATTCTAAC 4621 CAAATAATGTTTATCTCATGATTGTAGCTGATTCAACATTCAAAAATTGGCCTGGTGCAG 4681 TAGCTCAGGCCTGTAATCCCAACATTTTAGGAGGCTGAGGCAGGAAGATCTCTTGAGCCC 4741 AGGATTTCAAGACCAGCCTGGGCAACATAGTCAGACTGGTCTTTACTGGGGGGAAAAAAA 4801 TCAGTCTGTGTAATTCACCACATTAACAAAGGGAAACATAAAAACCCTATGATCATTTCA 4861 ACAGATGTAGCAAAAGCAGTTAATGATATTCAACACATATGCATGATTACAAACCAACCA 4921 ACCTCCTAGCAAACTAGGGAAAGGAAACTTAACCTAGTTTGATAACAGGGCGTCCACAGT 4981 CGGAGTTCCACTAGCAGCATACATAATGGTAGAAAACTCAGTGCTGCCGGGCGCGGTGGC 5041 TCACGCCTGTAATGCCAGCACTTTGGGAGGCCTAGGCGGGCGGATCACGAGGTCAGGAGA 5101 TCGAGACTGTCCTGACTAGCATGCTGAAACCCCGTCTCTACTAAAAATACAAAAACAAAA 5161 AATTAGCCGGGCATGGTGGCGGGCGCCTATAGTCCCAGCTACTCGGGAGGCTGAGGCGAG 5221 AGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGAGCCTAGATCGTGCCACTGCACTCCA 5281 GCCTGGGTGACAGAGTGAGACTTCGTCTCAAAAAAAAAAAAGAAAAGAAA 5341 ACTCAACGCTTTTTCCTCTAAGATCAGGAACTAGAAAAGGATTTGACTCTCACAACGTTG 5401 ATACCATACTGGAGGTTTTAACCAGGCAAGAAAAAGAAATAATGAGGGCCGGGTGCGGTG 5461 GCTCAGGCCTGTAATCCCAGCACTTTGGGAAGCCGAGACGGGTGGATCACGAGGTCAGGA 5521 GATCGAGACCATCCTGGCTAACACGGTGAAACCCTGTCTCTACTAAATATACAAAAAATT 5581 AGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAA 5641 TGGCGTGAACTCAGGGGGCGGAGCTTGCAGTGAGCTGAGATCGAGCCACTGCACTCCAGC 5701 CTGGGCGACAGAGCAAGACTGTGTCTCAAAAAAAAAAAAAGAAAAAGAAATAATGATTAG 5761 TGGCCCGATGTCTCACGCCTATAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCAC 5821 CTGAGGTCTGGAGTTGGAGACCAGCCTGACAAAGATGGTGAAACCTCGTCTCTATTAAAA 5881 TATTAAAAAAATAGCCAGGCGTTGGCCGGGTACAGTGGCTCATGCCTGTAATCCCAGCAC 5941 TTTGGGAGGCCGAGGTGGGTGGATCACCTGAGGTCAGGAGTTCAACACCAGCCTGGCCAA 6001 CATGGTGAAACCCCATCTCTACTAAAAATACAAAAATTAGCCGGGCGTAGTGGCGGGCGC 6061 CTGTAATCCCAGCTACTTGGGAGGCTTAGGCAGGAGAATCGCTTGAACCTGGGAGGCGGA 6121 GGTTGTAGTGAGCCGAGATTGCACCATTGCACTCCAGCCTGGGTGACAAAAGCAAAAACT 6181 CCGTCTCAAAAAAAAAAGAATTAGCCAGGGGTAGTGGTGAACGCCTGTAGTCCCAGCTAC 6241 TCAGGAGGCAGAGGCAGGAGAATCACTTGAACCCAGGAGGCAGAGGTTGCAGTGAGCCGA 6301 GATTGTCCCATTGCACTCCAGCCTAGGCGACAAGAGCAAAATTCCATGTCAAAAAAAAAA 6361 AAAAAAAAGGAAAGAAAAAAAATAACGATTAGAAAGGAAGAAATAAAACACATTCACAGC 6421 CAGTATGATTCTATACATACATGTCCTAATGGGGCCAGGCGTGGTGGCTCATGCCTGTAA 6481 TCCTAGCACTTTTAGGAGGCTGAGGCAGGTGGCTTCCCTGGGACCAGCCTGGCCAACATG 6541 GTGAAACCCCAACTCTAATAAAAATACAAAAAATCAGCCAGGCGTGGTGACGGGCACCTC 6601 TAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATTGCTTGGACCTGGGAGGCAGAGGT 6661 TGCAGTGAGCCGAGATCGCGCTATTGCACTCCAGCCTGGGCAACAAGAGTGAAACTCCGG 6721 CAGGGTGTGGTGGCTTACGCCTGTAATCCCAGCACTTCGGGAGGCTGAGGCAGGCCGATC 6781 ACCTGAGGTCAGGAGTTTGAGACCAACCTAACATGGTGAAACCCCGTCTCTACTAAAAAT 6841 ACAAGAATTAGCTGGGTGTAGTGGTGGGCGCCTGTAATCCCAGCTACTTGGGAGGCTGAG 6901 ACAGAAGAATTGCTTGAACCCAGGAGGTGGAGGTTGCAGTGAGCTGAGATCATGCCATTG 6961 CACACCACGCCGGGCAACAGAGCGAGATTCCGTCTCAAAAAAAAAAAAAAAGAGTGAAAC 7021 TCTATCTCAAAAAAAAAAAAAAGTCCTAATGGAAAATCCATAAAAAGCTACCAAAACTAA 7081 TAAATAAATATAGCAGGGTTGCAGGTTACAGGGCAATATAGTTATCCCTCTATCTGTAGG 7141 GGCTTGGTTCTGGGACTCCTCACACACCAAACCCACAGATGTCTAAGTCCCATATATAAG 7201 ACGGTATAGTATTTGGATTTAACCTACACATATCCTCCCATATAGTTTAAATTATCTCTA 7261 GATTACTTACATTACCCCCATACAATGAAAATGCTAATGTACATGCAAGTATGTATGTAA 7321 GTACTTGTACTATATTGTTTAGGGAATCACTGGACATATAGGCCTTCAAGACTGATACCA 7381 GCAGCCACTGTTAAGATTCTGGTCAGGCCTGCCCCTGTTTGGGGTCTCAGTTGATCTCAT 7441 TGCCTTCCCACCCAGCCAAGGGCACCTGCATTTCTCTTGGCTCCCTGGCCATTTGGAAGG 7501 CCTAGTTCAGCCTGGCACATTTGTATCCTGGCCCACTGATGCTGGTACCCCTGGGAAGGT 7561 CCTGCTCTGAAAAACACGGAGATTTTAGTTGCTACTGAAGATTTGAGAGATAAAGACAGG 7621 GAGACCTGTCTGTAGACCTGTGTCCCTCCAAGTGGGATTGAGACTTTGGGCCCCCCATTT 7681 CAGGACAGCACCTCCTGGCCTGTTGACTGAATAGATCCCTGAAGGAGGTGTACTTGCATT 7741 AATGGAGTGGGGGTGGGAGCAGTACCACAGATCCGCACTAACAATCACACAGTTCTCTCT 7801 AGAATAATAATATAGAACAAGTGAAATAGAACAATTGCAGAAAGAGCTAACCTTTGTTGA 7861 GCTCTTACTGTGTGCCCAGCACTTTCCTCAACTCTACATTTCCCATAATACACAGAGTAC 7921 TAGGTAGGCCAGGCTTGGTGGCTCACGCCTGTAATCCCAGCACTTTAGGAGGCCAAGGGG 7981 GGTGGATCACCTGAGGTCGGGAGTTCAAGACCAGCCTGACCAACATGGTGAAACCCCGTC 8041 TCTACTAGAAGTACAAAATTAGCCAGGTGTGGTGGCACATGCTTGTAGTCCTAGCTACTC 8101 AGCAGGCTGAGGCAGGAGAATCATTTGAATCCGGGAGGAGGTTGCAGTAAGCGGAGATAG 8161 TGCCACTGTACTCCAGCCTGGGCAATAAGAGCTGAGACTCCGTCTCAAAATAAAATAAAA 8221 TAAAATAAAAAAAGAAAAGAGCCTGCCATTAAAGGAGCTG 8281 TTTGGTAGGGGATGTTTTGTCAGTGCAAACAACAGAAAAGTGGGCTGGGCACAGTGGTTC 8341 ATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCGGATCACCTGAAGTTGGGAGT 8401 TCAAGACCAGCCTGACCAATATGGAGAAACCCCGTCTCTACTAAAAATACAAAATTAGCC 8461 GGGCGCAGTGGCGCATGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGC 8521 TTGAACCTGGGAGGCAGAGGTTGCGGTGAGCCGAGATCGCACCATTGCACTCCAGCCTGG 8581 ACGAGAGCAAAACTCTGTCTCAAAAAAAAAAAAAAACAGAAAAGTGTAACAAACACTTAC 8641 AGTAGGCATGTTTCTTAGCAAATCTGATGACAAATTTGGCATAAAGAAAGAGAGCATCCC 8701 TGAAAAAAAAAAAAAGAAAAAGAAAGAGAGCATCCTGCCTGGGCAACATAGTGAAACCCT 8761 GCCTCTACAAAAAAACTCAAAAATTGGCCGGGTGCAGTGGCTCACACCTGTAATCCCAGC 8821 ACTTTGGGAGTCGGAGGCGGGAGGATCACCTGAGGTCAGGAGTTCGAAACCAGCCTGGCC 8881 AACATGGCAAAACCCCATCTCTACTAAAAATACAAAAAATTAATCAGGCGCATTGGTGGG 8941 CGCCTGTAATCCCAGCTACTCAGGAAGTTGAGGCAAGAGGATCGCTTGAATCTGGGAGGT 9001 GGAGGTTACAGTGAGTCGAGATCACACCACTGCACTCTAGCCTGGGTGACAGGGCGAGAC 9061 TCCGTCTCCAAAAAAAAAAAGAAAAAGAAAAAGACTAAAAAATTAGCCAGGCAGGCCTCT 9121 GTGGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCACTGAGCCCAGGAGTCCGAGGC 9181 TGTAGTGAGCCATGATTGCACCACTGTACCCTAGCTTGGGCAACAAAGCAAGACCCTGCC 9241 TCAAAAGAAAAAAGAAAGAAAGAAAGAACATGGCGGGCCAGGCACAGTGGCTCACACCTG 9301 TAATCCCAGCGCTTTGAGAGGCCGAGGCAGGTGGATCACAAGGTCAGGAGTTCCACACCA 9361 GCCTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATCAGCCAGGCATG 9421 GTGGCAGGGGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTTGAAAC 9481 CAGAAGGCAGAGGTTGCAGTGAGCCTAGACTGCACCACTGCACTCCAGCCTGGGCGAAAA 9541 GAGCCAAACTCCATCTCAAAAAACAAACAAAAAAACAAAACAAAAGAAAACATGGCAAAG 9601 CCTTTGAAAGCTTGTCTGGGAGAAGGTGCGATGATAGTTGCATAACTTCGTGCAAGATGC 9661 TGGTCCACACAGGGGCTGCCCCTTGCTCTTTCTCGCTCTCTTAACCTCTCATATAACAGG 9721 CTTGTGTGTTATTCACATTTATTGAGCCCAAGCAGGTGCAAGGCATTGTGATCTAATACT 9781 TTGGTCAGCAAGACAACAAGATAGATCACTGCCCTGCCCTTAGGAAGTGTATATGCTATT 9841 AGAGGAAACAGATAAAATAAACAAGGAAAAGTATCAGACAATGTAAGTGCTATGAGAATG 9901 CAAATGAGGTGATGTGAATTAAAATAGGATGACTTAAAGTCTGCACGGGAAGGAGCCTAC 9961 CCCCATGTTCCTGGCTAGCCAAGGAACCACCAGTTGATTAGCAGAGAAGGGCAGCCAGTC 10021 TAGCTAGAGCTTTTGGGGAAGAGGGAGTGGTTGTTAAGAGATGAGATTAAAGAAGCCGAG 10081 ACGGGCCATTCGTGAGGGGTTTGTAATGCAGGGCTGAGGAGTGTCCGAAGAGAATGGGCA 10141 GGTGAGCGGTGAGACAGTTGTTCTTCCAGAAGCTTTGCAGTGAAAGGAATCAAAGAAATG 10201 GAGCCGTGTATCAGGTGGGGAAGGGTGGGGGCCAAGGGGGTGTCCTTCCCCATACAGAGA 10261 TTGCAGGCTGAGAATGACTATATCCTTGTTAACAGGAGGTGGGAGCAGGGCACGGTAGCT 10321 CACACCTGTAATCTTGGCACTTTAGGAGGCTGAGGCGGGCCGATCACCTGAAGTAAGGAG 10381 TTCGAGACCAGCCTGGCCAACATGCAAAGCCCTGTCTCTACTAAAAATACAAAAATTAGC 10441 TGGGTGTGGTGGTACTCGCCTGTAATCCCAGCTACTCGGGAGACTGAGGCAGGAGAATGG 10501 CTTGAACCCGGAAGGTAGAGGTTGCAGTGAGCTGAGATCATGCCACTGTGCTCCAGCCTA 10561 GGTGACAGAGAGAGACTCCATCTCAAAAAAAAAAAAAAAATACAGGAAGGGAGTTGGGAA 10621 TAGGGTGCACATTTAGGAAGTCTTGGGGATTTAGTGGTGGGAAGGTTGGAAGTCCCTCTC 10681 TGATTGTCTTTTCCTCAAAGAAGTGCATGGCTGGTGAGGGGTGGGGCAGGAGTGCTTGGG 10741 TTGTGGTGAAACATTGGAAGAGAGAATGTGAAGCAGCCATTCTTTTCCTGCTCCACAGGA 10801 AGCCGAGCTGTCTCAGACACTGGCATGGTGTTGGGGGAGGGGGTTCCTTCTCTGCAGGCC 10861 CAGGTGACCCAGGGTTGGAAGTGTCTCATGCTGGATCCCCACTTTTCCTCTTGCAGCAGC 10921 CAGACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC 10981 CCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTGTGAGTGGATCCATTGGAAGGG 11041 CAGGCCCACCACCCCCACCCCAACCCCAGCCCCCTAGCAGAGACCTGTGGGAAGCGAAAA 11101 TTCCATGGGACTGACTTTCTGCTCTTGTCTTTCAGACTTCCTGAAAACAACGTTCTGGTA 11161 AGGACAAGGGTTGGGCTGGGGACCTGGAGGGCTGGGGACCTGGAGGGCTGGGGGGCTGGG 11221 GGGCTGAGGACCTGGTCCTCTGACTGCTCTTTTCACCCATCTACAGTCCCCCTTGCCGTC 11281 CCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGA 11341 CCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACC 11401 AGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGT 11461 CCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGG 11521 GACAGCCAAGTCTGTGACTTGCACGGTCAGTTGCCCTGAGGGGCTGGCTTCCATGAGACT 11581 TCAATGCCTGGCCGTATCCCCCTGCATTTCTTTTGTTTGGAACTTTGGGATTCCTCTTCA 11641 CCCTTTGGCTTCCTGTCAGTGTTTTTTTATAGTTTACCCACTTAATGTGTGATCTCTGAC 11701 TCCTGTCCCAAAGTTGAATATTCCCCCCTTGAATTTGGGCTTTTATCCATCCCATCACAC 11761 CCTCAGCATCTCTCCTGGGGATGCAGAACTTTTCTTTTTCTTCATCCACGTGTATTCCTT 11821 GGCTTTTGAAAATAAGCTCCTGACCAGGCTTGGTGGCTCACACCTGCAATCCCAGCACTC 11881 TCAAAGAGGCCAAGGCAGGCAGATCACCTGAGCCCAGGAGTTCAAGACCAGCCTGGGTAA 11941 CATGATGAAACCTCGTCTCTACAAAAAAATACAAAAAATTAGCCAGGCATGGTGGTGCAC 12001 ACCTATAGTCCCAGCCACTTAGGAGGCTGAGGTGGGAAGATCACTTGAGGCCAGGAGATG 12061 GAGGCTGCAGTGAGCTGTGATCACACCACTGTGCTCCAGCCTGAGTGACAGAGCAAGACC 12121 CTATCTCAAAAAAAAAAAAAAAAAAGAAAAGCTCCTGAGGTGTAGACGCCAACTCTCTCT 12181 AGCTCGCTAGTGGGTTGCAGGAGGTGCTTACGCATGTTTGTTTCTTTGCTGCCGTCTTCC 12241 AGTTGCTTTATCTGTTCACTTGTGCCCTGACTTTCAACTCTGTCTCCTTCCTCTTCCTAC 12301 AGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGC 12361 TGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGC 12421 AGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATA 12481 GCGATGGTGAGCAGCTGGGGCTGGAGAGACGACAGGGCTGGTTGCCCAGGGTCCCCAGGC 12541 CTCTGATTCCTCACTGATTGCTCTTAGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGG 12601 AAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGG 12661 TGCCCTATGAGCCGCCTGAGGTCTGGTTTGCAACTGGGGTCTCTGGGAGGAGGGGTTAAG 12721 GGTGGTTGTCAGTGGCCCTCCAGGTGAGCAGTAGGGGGGCTTTCTCCTGCTGCTTATTTG 12781 ACCTCCCTATAACCCCATGAGATGTGCAAAGTAAATGGGTTTAACTATTGCACAGTTGAA 12841 AAAACTGAAGCTTACAGAGGCTAAGGGCCTCCCCTGCTTGGCTGGGCGCAGTGGCTCATG 12901 CCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGCGGATCACGAGGTTGGGAGATCGAG 12961 ACCATCCTGGCTAACGGTGAAACCCCGTCTCTACTGAAAAATACAAAAAAAAATTAGCCG 13021 GGCGTGGTGCTGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGAAGGAGAATGGCG 13081 TGAACCTGGGCGGTGGAGCTTGCAGTGAGCTGAGATCACGCCACTGCACTCCAGCCTGGG 13141 CGACAGAGCGAGATTCCATCTCAAAAAAAAAAAAAAAAGGCCTCCCCTGCTTGCCACAGG 13201 TCTCCCCAAGGCGCACTGGCCTCATCTTGGGCCTGTGTTATCTCCTAGGTTGGCTCTGAC 13261 TGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGG 13321 AGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCT 13381 GCACACTGGCCTGCTGTGCCCCAGCCTCTGCTTGCCTCTGACCCCTGGGCCCACCTCTTA 13441 CCGATTTCTTCCATACTACTACCCATCCACCTCTCATCACATCCCCGGCGGGGAATCTCC 13501 TTACTGCTCCCACTCAGTTTTCTTTTCTCTGGCTTTGGGACCTCTTAACCTGTGGCTTCT 13561 CCTCCACCTACCTGGAGCTGGAGCTTAGGCTCCAGAAAGGACAAGGGTGGTTGGGAGTAG 13621 ATGGAGCCTGGTTTTTTAAATGGGACAGGTAGGACCTGATTTCCTTACTGCCTCTTGCTT 13681 CTCTTTTCCTATCCTGAGTAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGT 13741 TTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGA 13801 GCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGGTAAGCAAGCAGGACAAGAAGC 13861 GGTGGAGGAGACCAAGGGTGCAGTTATGCCTCAGATTCACTTTTATCACCTTTCCTTGCC 13921 TCTTTCCTAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTG 13981 GATGGAGAATATTTCACCCTTCAGGTACTAAGTCTTGGGACCTCTTATCAAGTGGAAAGT 14041 TTCCAGTCTAACACTCAAAATGCCGTTTTCTTCTTGACTGTTTTACCTGCAATTGGGGCA 14101 TTTGCCATCAGGGGGCAGTGATGCCTCAAAGACAATGGCTCCTGGTTGTAGCTAACTAAC 14161 TTCAGAACACCAACTTATACCATAATATATATTTTAAAGGACCAGACCAGCTTTCAAAAA 14221 GAAAATTGTTAAAGAGAGCATGAAAATGGTTCTATGACTTTGCCTGATACAGATGCTACT 14281 TGACTTACGATGGTGTTACTTCCTGATAAACTCGTCGTAAGTTGAAAATATTGTAAGTTG 14341 AAAATGGATTTAATACACCTAATCTAAGGAACATCATAGCTTAGCCTAGCCTGCTTTTTT 14401 TTTTTTTTTTTTTGGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCGGG 14461 ATCTCGGCTCACTGCAACCTCCGCCTTCTGGGTTCAAGCGATTCTCCTGCCTCAGCCCAC 14521 TGAGTAGCTGGGATTACAGGCACCTGCCCCGACGCCCAGCTAATTTTTTGTTATTTATTT 14581 ATTTTTTTTTTTAGTAGAGATGAGGTTTCACCATGTTGGCCAGGCTAGTCTCGAACTCCT 14641 GACCTTGTGATCTGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACC 14701 GCACCCGGCCTGCCTAGCCTACTTTTATTTTATTTTTAATGGAGACAGCATCTTGCTCTG 14761 TTGCCCAGGCTGGATTACAGTGATGTGATCATAGCTCATTATACCCTCCTGGGCTCAAGC 14821 AATCCCCCTAACTCTGCCTCCCCAGTAGCTAGGACCACAGGCATACACCACCATACCCAG 14881 CTAATTTTTAAAATTTTTTGTAGATAGATAGAGTCTCACTATGTTGCCCAGGCTGGTCTC 14941 TAGCCTACTTTTTTGAGACAAGGTCTTGCTCTGTCACCCAGGCTGGATAGAGTGCAGTAG 15001 TGCAGTCACAGCTCACTGCAGCCTCCACCTCCCAGGCTCCATCCATCCTCCCAGCTCAGC 15061 CTCCCAAGTTGCTTCAACTACAGGCCTGCACCACCATGCCTGGCTAATTTTTATTTATTT 15121 ATTTTTATTTTATTTTATTTTATTTTTTTGAGACTCAGTCTCACTCTGTCGCCCAGGCTG 15181 GAGTGCAGTGGCATGATCTCGGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATTCT 15241 CCTGCCTCAGCCTCCCGAATAGCTAGGACTACAAGCGCCTGCTACCACGCCCAGCTAATT 15301 TTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTTCTG 15361 ACCATGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCA 15421 CGCCCGGCTAATTTTTATTTATTTATTTAAAGACAGAGTCTCACTCTGTCACTCAGGCTA 15481 GAGTGCAGTGGCACCATCTCAGCTCACTGCAGCCTTGACCTCCCTGGGCTCCGGTGATTT 15541 CACCCTCCCAAGTAGCTAGGACTACAGGCACATGCCACGACACCCAGCTAATTTTTTATT 15601 TTCTGTGAAGTCAAGGTCTTGCTACGTTGCCCATGCTGGTATCAAACCCCTGGGCTCAAT 15661 CAATCCTTCCACCTCAGCCTCCCCAAGTATTGGGGTTACAGGCATGAGCTACCACACTCA 15721 GCCCTAGCCTACTTGAAACGTGTTCAGAGCATTTAAGTTACCCTACAGTTGGGCAAAGTC 15781 ATCTAACACAAAGCCCTTTTTATAGTAATAAAATGTTGTATATCTCATGTGATTTATTGA 15841 ATATTGTTACTGAAAGTGAGAAACAGCATGGTTGCATGAAAGGAGGCACAGTCGAGCCAG 15901 GCACAGCCTGGGCGCAGAGCGAGACTCAAAAAAAGAAAAGGCCAGGCGCACTGGCTCACG 15961 CCTGTAATCCCAGCATTTCGGGAGGCTGAGGCGGGTGGATCACCTGAGGTCAGGAGTTCA 16021 AGACCAGCCTAGCCAACATGGTGAAACCCCGTCTCTACTAAAATACAAAAATTAACCGGG 16081 CGTGATGGCAGGTGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTG 16141 AACCAGGAGGCGGAGGTTGCAGGGAGCCAAGATGGCGCCACTGCACTCCAGCCTGGGCGA 16201 TAGAGTGAGACTCCGTCTCAGAAAAAAAAGAAAAGAAACGAGGCACAGTCGCATGCACAT 16261 GTAGTCCCAGTTACTTGAGAGGCTAAGGCAGGAGGATCTCTTGAGCCCAAGAGTTTGAGT 16321 CCAGCCTGAACAACATAGCAAGACATCATCTCTAAAATTTAAAAAAGGGCCGGGCACAGT 16381 GGCTCACACCTGTAATCCCAGCACTTTGGGAGGTGGAGGTGGGTAGATCACCTGACGTCA 16441 GGAGTTGGAAACCAGCCTGGCTAACATGGTGAAGCCCCATCTCTACTAAAAACACAAAAA 16501 TTAGCCAGGTGTGGTAGCACACGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCACAAG 16561 AATCACTTGAACCCCAGAGGCGGAGATTGCAATCAGCCAAGATTGCACCATTGCACTCCC 16621 GCCTGGGCAACAGAGTGAGACCCCATCTCAAAATAAATAAATAAATATTTTTAAAAGTCA 16681 GCTGTATAGGTACTTGAAGTGCAGTTTCTACTAAATGCATGTTGCTTTTGTACCGTCATA 16741 AAGTCAAACAATTGTAACTTGAACCATCTTTTAACTCAGGTACTGTGTATATACTTACTT 16801 CTCCCCCTCCTCTGTTGCTGCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGC 16861 TGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGG 16921 CTCACTCCAGGTGAGTGACCTCAGCCCCTTCCTGGCCCTACTCCCCTGCCTTCCTAGGTT 16981 GGAAAGCCATAGGATTCCATTCTCATCCTGCCTTCATGGTCAAAGGCAGCTGACCCCATC 17041 TCATTGGGTCCCAGCCCTGCACAGACATTTTTTTAGTCTTCCTCCGGTTGAATCCTATAA 17101 CCACATTCTTGCCTCAGTGTATCCACAGAACATCCAAACCCAGGGACGAGTGTGGATACT 17161 TCTTTGCCATTCTCCGCAACTCCCAGCCCAGAGCTGGAGGGTCTCAAGGAGGGGCCTAAT 17221 AATTGTGTAATACTGAATACAGCCAGAGTTTCAGGTCATATACTCAGCCCTGCCATGCAC 17281 CGGCAGGTCCTAGGTGACCCCCGTCAAACTCAGTTTCCTTATATATAAAATGGGGTAAGG 17341 GGGCCGGGCGCAGTGGCTCACGAATCCCACACTCTGGGAGGCCAAGGCGAGTGGATCACC 17401 TGAGGTCGGGAGTTTGAGCCCAGCCTGACCAACATGGAGAAACCCCATCTCTACTAAAAA 17461 TACAAAAGTAGCCGGGCGTGGTGATGCATGCCTGTAATCCCAGCTACCTACTCGGGAGGC 17521 TGAGGCAGGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCGGTGAGCTGAGATCTCACC 17581 ATTACACTCCAGCCTGGGCAACAAGAGTGAAACTCCGTCTCAAAAAAGATAAATAAAGTA 17641 AAATGGGGTAAGGGAAGATTACGAGACTAATACACACTAATACTCTGAGGTGCTCAGTAA 17701 ACATATTTGCATGGGGTGTGGCCACCATCTTGATTTGAATTCCCGTTGTCCCAGCCTTAG 17761 GCCCTTCAAAGCATTGGTCAGGGAAAAGGGGCACAGACCCTCTCACTCATGTGATGTCAT 17821 CTCTCCTCCCTGCTTCTGTCTCCTACAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCT 17881 CCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGACATTCTCCAC 17941 TTCTTGTTCCCCACTGACAGCCTCCCACCCCCATCTCTCCCTCCCCTGCCATTTTGGGTT 18001 TTGGGTCTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATT 18061 TGCTTTGTCCCGGGGCTCCACTGAACAAGTTGGCCTGCACTGGTGTTTTGTTGTGGGGAG 18121 GAGGATGGGGAGTAGGACATACCAGCTTAGATTTTAAGGTTTTTACTGTGAGGGATGTTT 18181 GGGAGATGTAAGAAATGTTCTTGCAGTTAAGGGTTAGTTTACAATCAGCCACATTCTAGG 18241 TAGGGGCCCACTTCACCGTACTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTA 18301 ACTTCAAGGCCCATATCTGTGAAATGCTGGCATTTGCACCTACCTCACAGAGTGCATTGT 18361 GAGGGTTAATGAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGGGGTCTA 18421 GAACTTGACCCCCTTGAGGGTGCTTGTTCCCTCTCCCTGTTGGTCGGTGGGTTGGTAGTT 18481 TCTACAGTTGGGCAGCTGGTTAGGTAGAGGGAGTTGTCAAGTCTCTGCTGGCCCAGCCAA 18541 ACCCTGTCTGACAACCTCTTGGTGAACCTTAGTACCTAAAAGGAAATCTCACCCCATCCC 18601 ACACCCTGGAGGATTTCATCTCTTGTATATGATGATCTGGATCCACCAAGACTTGTTTTA 18661 TGCTCAGGGTCAATTTCTTTTTTCTTTTTTTTTTTTTTTTTTCTTTTTCTTTGAGACTGG 18721 GTCTCGCTTTGTTGCCCAGGCTGGAGTGGAGTGGCGTGATCTTGGCTTACTGCAGCCTTT 18781 GCCTCCCCGGCTCGAGCAGTCCTGCCTCAGCCTCCGGAGTAGCTGGGACCACAGGTTCAT 18841 GCCACCATGGCCAGCCAACTTTTGCATGTTTTGTAGAGATGGGGTCTCACAGTGTTGCCC 18901 AGGCTGGTCTCAAACTCCTGGGCTCAGGCGATCCACCTGTCTCAGCCTCCCAGAGTGCTG 18961 GGATTACAATTGTGAGCCACCACGTCCAGCTGGAAGGGTCAACATCTTTTACATTCTGCA 19021 AGCACATCTGCATTTTCACCCCACCCTTCCCCTCCTTCTCCCTTTTTATATCCCATTTTT 19081 ATATCGATCTCTTATTTTACAATAAAACTTTGCTGCCACCTGTGTGTCTGAGGGGTGAAC 19141 GCCAGTGCAGGCTACTGGGGTCAGCAGGTGCAGGGGTGAGTGAGGAGGTGCTGGGAAGCA 19201 GCCACCTGAGTCTGCAATGAGTGTGGGCTGGGGGGCCCAGTGCCCGGGTTCCGGGAGGGG 19261 AACAAAGGCTGGAGACTGGGTCAGTCTGCGGGCTGCATGACAACAAGGGAGGGGGTGGCT 19321 CCATTCATAACTCAGGAACCAACCGTCCCTCCTCCCCTCCGGCCACGGCTGGCACAAGGT 19381 TCTCTCCCTCCCCTGCTTCTAGGACTGGGCTGCTTCCCCCTCGGCAGCCTCTCACCAAGG 19441 ATTACGGGATTTAAATGTCTGATTTAGCAAGGCTGAGCCTCCAGGGTGGCCATCTGCTCC 19501 ATCAGAAAGTGGCAGGATACCTGGGTTCCCAAGGGGAACAGGGGTGGGTGCTACTGGATG 19561 GAGAGAGGCCAGTGGGAGGCCTGCTAGCCAGGGTCCCAGGAAAGTGGGGGCAGCTAAGGT 19621 AAGAGTAGGGGTGTGGGGCTAGGTCCTTCCCAGCATCCCCTCATCCTGGGCCTCATGCCA 19681 GGTAGCTGAATGAATTGAAGCTTTAAACTCTGCCAGGAAAACCTTTCAAAGGGCTTCTTG 19741 GGATAGGGAGGAGAGTCGGGTTGAGGAGCTCAGTACTGCCTGCCCATGCTCCTCAGGGCT 19801 GCTGGCTCCCAGGGAGGGGGGCTGGGAGCAGGCAGGCTCTTCCCCATCACCCACTGCTCT 19861 CTTGGAGCCAGTGCTTGAAGGGGCAGTCAGACATGGCTTGCCCTTCCTCCTCCCTGGTGG 19921 TGGAGATGGGTGTTAGGGTCCAGTGGGTGCTACTGTCCAGGGGGGCTTCTGGGGCCACCA 19981 GCCTGTCAGCTCATCAACCAGGCTGAAGGTGCAAGCAGGAGCCCCTTGCCTTGCCCCAAG 20041 GATCCCAGACAGCTATGAAGCCACCAGCCTTCCTGACCTCAAGACCACCTTTTTTTTTTC 20101 TCTTTCTTACTAGGGAATGCCAAACACTCTCCCCAGGAGATCCAGACCCGCCTCTTTCAG 20161 AGACTTTTAACTTAAACATCTGTCCCTACCCAGCAGGCAAACTAGAGCTCCTGAAGCTCA 20221 GTCCCTGTCCTTGCCTCTGTAGACAGGTCACCTTGATGAGCTTCCTTTTTTTTTTTTTAA 20281 TTTTTTTTTATTTTAGGCTTTATTGGGGCATAATTGATCCCCCAAAATTGCATACATTCA 20341 AGGTATGCAGTGTGATGATTTGATATGGGGGTATATTGTGAAACCATTACCACAATCAAA 20401 TTAATCAGCACGTCCATCATCACACACAGTTACCATTTGTGTGTGTGCACGTGTGTTCAC 20461 CTACGACGAGGACACTTGGACCTACTCTGCAGATCTCAAGTAAACAGAAAATCTCCCTTT 20521 TTGACAACCATCCTCCACCCTTTCAATCCCAACCTTTTCCTAGATTATGTCCCTAGCTCT 20581 GTTTTTATTTCTGCTGTGCTGCTTCAGATCCATTCTGACTCTGCCAAACCCTTCTTTGTG 20641 AGCTGATAGATTGCTGGATTGAGAATTACAGCTGGGCGCGGTGGCTCACGCCTGTAATCC 20701 CAACACTGTGGGAGGCCAAGGCCGGCGGATCACTTGAGGTCAGGAGTTGGAGACCAGCCT 20761 GACCAACAAGATGAAACCCCATCTCTACTAAAAATACAAAATTAGCTGGGCATGGTGGTG 20821 CACGCCTGTAATCTCATCTTCTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCGGGAG 20881 GTGGAGGTTGCAGTGAGCCAAGATCCTGCCATTGCACTCCAGCCTGGGCAACAACAGTGA 20941 AGCTCCATCTCAAAACACACAAAAAAAAGAAGTACAAAGTCTGAGACTTCAGGCCAGCTC 21001 TGCTACACTATATACTCTAACCTCTCTGGTCCTACTTGGTGACTTCTTTCCCTCTGGTCG 21061 TGTTCAAGTTCCCGTCCCATCCAGTCAAGCAGGTACTCATTGGTACCTTACCCTGTGCCA 21121 GGAGCTGTTCTAGGCCCTGGAAACCTATGGCAGACATGTTCCCTACCCTCCCACTCAAAG 21181 AGCCCAGGCCTTATCCTAATGAGATCTGAAATCAAATCTCCCAATTTCCTCATGGCTTCA 21241 GTCTAAACTTGTAATTCACAACCTTAAATCAATATGTTCTATTTTTTTATTTAGAAAACA 21301 TTTCCGGCCAGGCACGGTGGATCACACCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAG 21361 GAGAATCGCTTGAACCCAGGAGGCAGAGGGTTGCAGTGAGCCGAGATTGCGCCATTGCAC 21421 TCTAGCCTGGGCAACAGAGCAAGACTCCATCTCAAAAAAGAAAAAAAAATGGAAGAAAAA 21481 AAAATTTCCCCCTCATTTTAGGAACACGAGGTCTCCAAATCTAAAATTCGTACTCTGAGG 21541 AGATTGAATAGCCTTAAATGCTTTCATCATTAAAAAGAAAAGAAAGGAACCTGGTATGCA 21601 TCCTAAAAATGAAAAATATACCTACCTGTAATCCCAGCACACAGCACATTGGGAGGCTAA 21661 AGCAGGAGGATAACTTGAGGCCAGGAGTTTCAGATCAGCCTGGGCAACATAGCAACACCC 21721 CATTTCTTTTTCTTTTCTTTTTTTTTTGGAGACACAGTCTCGCTCTGTTACTCAGGCTGG 21781 AGTGCAGTGGCTCAATCTCAGCTCACTGCAAGCTCTGCCTCCCAGGTTCATGCCATTCTC 21841 CTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCGCCACCACGCCTGGCTAATTT 21901 TTTGTATTTTTAGTAGAGACAGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTG 21961 ACCTCGTGATCCGCCAGCCTTGGCCTCCTAAAGTACTGGGATTACAGGCGTGAGCCACTG 22021 CGCCTGGCCACAACACCCCATTTCTATTTTAATAAAATAAAATACTGTGAAAAACATTTA 22081 CAATTTTTAAATTTTAATTTTAAAATTAAACTTATATTTATTCATTTGTGTGTGTGGGTT 22141 TTTTTTTTTTTTTTTTTTTGCTTTTTTTTTGAGATGGAGTGTCACTCTGTCACCCAGGCT 22201 GGAGTGCAGTGGCGTGATCTCTGCCTCCCGGTTCAAGTGATTCTCCTGCCATAGCCTCCC 22261 AAGTAGCTGGGACTACAGGTACACGCCACCACGCCGGGTTAATTTTTGTATTTTTAGTAG 22321 AGACAGGATTTCACTGTGTCGCCAGGCTAGCCTCGAACTCCTGACCTCAGGTGATTCGCC 22381 CACCTTGGCCTCCCAAAGTGCTGTGATTACAAGCGTGAGCCACCGTGCCCAGCCCAAAGT 22441 TGGTTTTAATAGCAGAAAATCTATCAACATAATTCAATATATTAAATTTAGAAAGAAAAA 22501 TTATCTATCATATCAACAGATACTGAAAGGAATTTGATTAAATTTCAGTAGCCATTTCCT 22561 TAAAAAAGAAAACACTTTAACACAGTAATAGACTGATAATGGAATACCAATTTTCCTAAT 22621 AAGTTAAACATTAAGATAATTTCAATTAAGGTCAAGAGCTGGGCCAGGTGCAGTGGCTCA 22681 CACCTGTAATCCCAACACTTTGGAGGCCAAGGTGGGTGGATCACCTGAGGTCAGGAGTGG 22741 AGACCAGCCTGGCTGACAATAGTGAAATCCTGCCTCTACTAAAAACACAAAAAATTAGCT 22801 GGGCATGGTGGTGGGCACCTATAATCCCAGCTACTGGGAAGGCTGAGACAGGAGAATTGC 22861 TTGAACCTGGGAGGCGGAGGTTGCAGTGAGCAAAGATCACACCATTGCACTCCAGCCTGG 22921 GCGACAGAGCCAGAGTCAGTCTCAAAAAAAAAAAGAGGTGGCCACACCTATAATCCAAAC 22981 ATTTTGTGAGGCCAAGGCAGGAGAATTGCTTCAGGCCAAGAGTTGAACACCTCGTCAACA 23041 TAGCCAGACCTCTCTCTAGATAGATAGATAGATGATAGATAGAGAGATAGATAGATGATA 23101 GATAGAGAGATAGATAGATGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAG 23161 ATAGATAGATAGATAGATAATCTGGCCGGGTGTGGAGGCTCACGCCTGTAATCCCAGCAC 23221 TTTGGGAGGCTGAGGCGGGCAGATCACGAGGACAAGAGATTGAAACCATCCTGGCTAACA 23281 AGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGGCGGGTGTGGTGGCACGCGCC 23341 TGTAGTCCTAGCTATTCAGGAGGCTGAGACAGGAGAATTGCTTGAATCCGAAAGGCGGAG 23401 GTTGCAGCGAGCCGAGATCGTGCCACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCCA 23461 TCTCAAAATAAATAAATAAATAATCAAGAACAGTATAAGGGGCTGTATGGTGGCTCATGC 23521 CTGTGATCCCAGCACTTTGGGAGGCCAAGGTGGGAGGATCCCTTGAGACCAGCCCAGGCA 23581 ACAGAGAAAGACCCTGTCTCTATTTAAAAAAATTAAAAACTGGCCGGGCACGGTGGCTCA 23641 CGCCTGTAATTCCAGCGCTTGGGAGGCCAAGGCAGGCACATCAGGAGGTCAGGAGTTCGA 23701 GACCAGCCTGGCCAACGTGGTGAAACCCCGTCTCTACTAAAAATACAAAAAGTAGCTAGG 23761 CGTGGTGGCAGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTG 23821 AACCCAGGAGGCGGAGGTTGCAGTGGGCAAAGATCGTGCCATTGCACTCAGCCTGGGTGA 23881 CAGGGCAAGACTCCATCTCAAAATAAATAAACAAAGTAATTAATTAATTAAATTAAAAAC 23941 TGTGGGGATATAGACTTACTCTGGTTTTATTTTTTCTTTTCTTTTCTTTTCTTTTTTCTG 24001 AGACGGAGTCTCGCTCTGTTGCCCAGGCTGGAGTACAGTGGCGTGGTTTCTGTTCTCTGC 24061 AACCTCCACCTCCCGGATTCAAGCGATTCTCTTGCCTCAGCCTCTTGAATACCTGGAATT 24121 ACAGGTGCCTGCCACCACCCCCGGCTAATTTTTTGTATTTTTAGTAGAGACAGGGTTTCA 24181 CCATGTTGGCCAAGCTGGTCTCGAACTCCTGACCTCATGATCCACCCGCCTCTGCCTCCC 24241 AAAGCACTGAGACTACAGGAGTGAGCCACTGTGCCCAGCCTACTCTGGTTTTAGTGCATT 24301 CAAGAGGAACAAAAAAGGAAGAAAATCACTAGTAAATATACCTCTTTCTGGTTAGAGTGG 24361 ATGTTTGGAAATTATATATATATTATATTATATTATATATATTATATATATACACAAACA 24421 CGTACATACATGCACACACATATATGCCTTTTTGATTATAGGATAGTATACCAAAACTCA 24481 GAAATATTATGGAATTAACAGAATTTAGTAAGGCAGATAAGTAGTAGGTAGAAAAATATT 24541 AATTTTATCTTCCAGCAGAAGCACTGTGAAAAATTAGACAACAAGAAAACATTCCATTCA 24601 AAATAATGACAATAAGGCCGGGCATGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAG 24661 GCTGAGGCAGGAGGATCATCTGAGGTCAAGTTTGAGATCAGCCTGGCCAACATGGTGACA 24721 CCCTGTCTCTACTGAAAATACAAAAATCAGCCAGCTATGGTAGTGTAAGCCTGTAATTCC 24781 AGCTACTCGGGAGGTCGAAGCAGAAGAATCACTTGAACCCAGGAGGCAGAGATTGCAGTG 24841 AGCCAAGATCCTGCCAGTGCTTTCCAGCCTGGGCAACAGTGTGAGGCTCCATCTCAAAAA 24901 AAAAAAAAAAAAAAAGACAATAGCAATAAACATTAAGAAATGTGTAATAGGAATGGCACA 24961 CACAAAGAAGGAATGGCACAGAGCCTGTATGCAGAAGACCACAAACCCTTATTTAACGAC 25021 GTAAGCCAAGATCCAAAGAAAATGATAGATTCTCAGATGGGAAAACTAAAAAAATAAGAA 25081 AAATCAATTATCTCGAGATAAATATAATATAATGCAATTTCAATTAGAATCCCAAATTTT 25141 CATTGTGTGTGTGTGTGAGTTGGGTAAATTTATCATAAATGTATAGGAACGAGTAAGTGT 25201 CACTAGTTGTTTAAATAAATACTGGATTTGGGCCAGGCATGGTGGCTCACGCCTCTAATC 25261 CCAGCACTTTGGGAGACCGAGGCGGGCAGATCATGAGGTCAGGAGATCGAGACCATCTGG 25321 CCAACATAGTGAAAACTCGTCTCTACTAAAGATACAAAAAATTAGCTGGGCATGGTGGCA 25381 CGTGCCTGTAGTTCCAGCTACTCTGGAGGCTGAGGCAGGAGAGTTGCTTGAACCCGGGAG 25441 GTGGAGGTTGCAATGAGCCGAGATCCTGTCACTGCACTCCACCCTGGCGACAAAGTGAGA 25501 CTCCGTCTCTCTCTCTCTCTTTAGGCCAAGGCAGGTGGATCACCTGAGGTCAGGAGTTCA 25561 AGACAGCCTGGCCAACATAGCGAAATCCCATCTCTACTAAAAATACAAAAATTAGCCTGG 25621 CAGTGGTGGCCCACGCCTGTAATCCCAGCTACTAAGGGGGCTGAGGCAGGAGGATCTCTT 25681 AACCAGGGAGGAGGAGGTTGCAGTGAGCAGAGATTGTGCCACTGCACTCCAGCCTGTGCA 25741 ACAGAGTGAGACTCTGTCTC

Coding Regions

[0046] Exon 1 includes Seq. ID No. 1 base numbers: 1-162.

[0047] Exon 2 includes Seq. ID No. 1, base numbers: 10917-11018.

[0048] Exon 3 includes Seq. ID No. 1, base numbers: 11136-11157.

[0049] Exon 4 includes Seq. ID No. 1, base numbers: 11267-11545.

[0050] Exon 5 includes Seq. ID No. 1, base numbers: 12303-12486.

[0051] Exon 6 includes Seq. ID No. 1, base numbers: 12568-12680.

[0052] Exon 7 includes Seq. ID No. 1, base numbers: 13249-13358.

[0053] Exon 8 includes Seq. ID No. 1, base numbers: 13702-13838.

[0054] Exon 9 includes Seq. ID No. 1, base numbers: 13931-14004.

[0055] Exon 10 includes Seq. ID No. 1, base numbers: 16824-16930.

[0056] Exon 11 includes Seq. ID No. 1, base numbers: 17849-19137.

[0057] Table 1 includes the nucleotide sequences for Exons 1-11, which uses the base numbers with reference to Seq. ID No. 1 to determine the individual sequences.

TABLE-US-00002 TABLE1 Exonnucleotidesequences. Exon IDNo. Sequence 1 GTTTTCCCCTCCCATGTGCTCAAGACTGGCGCTAAAAGTTTTGAGCTTCTCAAAAGTCTA GAGCCACCGTCCAGGGAGCAGGTAGCTGCTGGGCTCCGGGGACACTTTGC GTTCGGGCTGGGAGCGTGCTTTCCACGACGGTGACACGCTTCCCTGGATTGG 2 CAGCCAGACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAG CGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACT 3 ACTTCCTGAAAACAACGTTCTG 4 TCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATT GAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCC AGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGG CCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACC AGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAG TCTGTGACTTGCACG 5 TACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGC TGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCC ATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCA CCATGAGCGCTGCTCAGATAGCGATG 6 GTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAG TATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGA GCCGCCTGAG 7 GTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCT GCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAG 8 TGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAG ACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCAC GAGCTGCCCCCAGGGAGCACTAAGCGAG 9 CACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTG GATGGAGAATATTTCACCCTTCAG 10 ATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTC AAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAG 11 CCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATG TTCAAGACAGAAGGGCCTGACTCAGACTGACATTCTCCACTTCTTGTTCC CCACTGACAGCCTCCCACCCCCATCTCTCCCTCCCCTGCCATTTTGGGTTTTGGGTCTTT GAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATT TGCTTTGTCCCGGGGCTCCACTGAACAAGTTGGCCTGCACTGGTGTTTTG TTGTGGGGAGGAGGATGGGGAGTAGGACATACCAGCTTAGATTTTAAGGT TTTTACTGTGAGGGATGTTTGGGAGATGTAAGAAATGTTCTTGCAGTTAA GGGTTAGTTTACAATCAGCCACATTCTAGGTAGGGGCCCACTTCACCGTA CTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTAACTTCAAGGCCCATATCTGT GAAATGCTGGCATTTGCACCTACCTCACAGAGTGCATTGTGAGGGTTAAT GAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGGGGTCTA GAACTTGACCCCCTTGAGGGTGCTTGTTCCCTCTCCCTGTTGGTCGGTGGGTTGGTAGTT TCTACAGTTGGGCAGCTGGTTAGGTAGAGGGAGTTGTCAAGTCTCTGCTG GCCCAGCCAAACCCTGTCTGACAACCTCTTGGTGAACCTTAGTACCTAAA AGGAAATCTCACCCCATCCCACACCCTGGAGGATTTCATCTCTTGTATAT GATGATCTGGATCCACCAAGACTTGTTTTATGCTCAGGGTCAATTTCTTTTTTCTTTTTT TTTTTTTTTTTTCTTTTTCTTTGAGACTGGGTCTCGCTTTGTTGCCCAGGCTGGAGTGGA GTGGCGTGATCTTGGCTTACTGCAGCCTTTGCCTCCCCGGCTCGAGCAGTCCTGCCTCAG CCTCCGGAGTAGCTGGGACCACAGGTTCATGCCACCATGGCCAGCCAACT TTTGCATGTTTTGTAGAGATGGGGTCTCACAGTGTTGCCCAGGCTGGTCTCAAACTCCTG GGCTCAGGCGATCCACCTGTCTCAGCCTCCCAGAGTGCTGGGATTACAAT TGTGAGCCACCACGTCCAGCTGGAAGGGTCAACATCTTTTACATTCTGCA AGCACATCTGCATTTTCACCCCACCCTTCCCCTCCTTCTCCCTTTTTATATCCCATTTTT ATATCGATCTCTTATTTTACAATAAAACTTTGCTGCCACCTGTGTGTCTGAGGGGTG
TP53 Knock Out in MCF7 Cells with CRISPR Cas9

[0058] TP53 knockout MCF7 cells were generated as previously published [1]. Human codon-optimized Streptococcus pyogenes wild type Cas9 (Cas9-2A-GFP) was obtained by Addgene (Cat. No. 44719). Chimeric guide RNA expression cassettes with different sgRNAs (sgRNA1: CCATTGTTCAATATCGTCCG; sgRNA2: GACGGAAACCGTAGCTGCCC; sgRNA3: TGGTTATAGGATTCAACCGG) were ordered as gBlocks. These gBlocks were amplified by PCR using primers: gBlock_Amplifying_F: 5-GTACAAAAAAGCAGGCTTTAAAGG-3 and gBlock_Amplifying R: 5-TAATGCCAACTTTGTACAAGAAAGC-3. The PCR product was purified by Agencourt Ampure XP PCR Purification beads per the manufacturer's protocol (Beckman Coulter). One microgram of Cas9 plasmid and 0.3 g of each gRNA gBlock (pair 1: sgRNA1 & sgRNA2; pair 2: sgRNA1 & sgRNA3) were cotransfected into MCF7 cells via Lipofectamine 3000 in a 6-well plate. Knockout cells created using the pair sgRNA1 & sgRNA2 were named KO5.6, and knockout cells created using the pair sgRNA1 & sgnRNA3 were named KO3.4. Knockout pools were cultured in 10 M Nutlin-3a (SelleckChem) for 2 months, changing nutlin-3a treated media every 3 days and passaging cells every 6 to 8 days. Isogenic clones were isolated from the knockout and wild type pools via limiting dilution in a 96-well plate and incubated at 37 C. in a CO2 incubator for 15 days. Wild type clones were named Parental (PR).

Sanger Sequencing

[0059] DNA was isolated from each MCF7 wild type pool, knockout pool, and single cell clone following the Agencourt DNAdvance genomic DNA isolation kit. MCF7 wild type and KO5.6 cells were PCR amplified using primers: TP53_exon_4_F: 5-CTGGTAAGGACAAGGGTTGG-3 and TP53_exon_4_R: 5-GCCAAAGGGTGAAGAGGAAT-3. MCF7 KO3.4 cells were PCR amplified using primers: TP53_exon_4_F and TP53_Woke_R: 5-ATTAGGCCCCTCCTTGAGAC-3. Products were sent to Eton Bioscience Inc. who purified the PCR products and performed Sanger Sequencing.

Western Blotting

[0060] Protein was extracted from cells using 2-Mercaptoethanol and Laemmli sample buffer (Bio Rad, Cat. No. 1610737). MCF7 wild type and knockout derivative proteins (10 g) were separated on 4-12% gradient polyacrylamide gels via SDS-PAGE and transferred to PVDF membranes (Millipore). Primary antibody dilutions were 1:300 for TP53 and 1:500 for -actin. Drug Screening

[0061] MCF7 wild type and knockout derivative pools were plated at a density of 5000 cells/well in polystyrene, flat-bottom 96-well plates. All 133 compounds from the NCI Approved oncology drugs set IV (Table 2, AOD-IV Drug) were dissolved in DMF or DMSO at 10 mM stocks. Cells were treated at concentrations from 156.25 nM to 10 M for 10 days. DMSO or DMF vehicle was used as a negative control. After 10 days, resazurin (Sigma, Cat. No. R7017) was added to each well and incubated at 37 C. in a dark CO2 incubator for 4 to 6 hours. A microplate reader took optical measurements (ex: 535 nm/em: 585 nm). Drugs showing larger Area under the curve (AUC) differences based on fluorescence values between MCF7 TP53 wild type and knockout cell lines were selected and further characterized, notably: oxaliplatin, 5-fluorouracil, and palbociclib.

Results

[0062] FIG. 1A illustrates an example knockout strategy/design and predicted deletion. FIG. 1B shows example targets of sgRNA1, 2, and 3 within TP53 of KO3.4 and KO5.6. sgRNA1 targets within exon 4, sgRNA2 targets downstream of sgRNA1 within exon 4, and sgNRA3 targets the intron after exon 10.

[0063] FIG. 2 illustrates an example gel shown using primers TP53_exon_4_F and TP53_Woke_R, only KO3.4 single cell clones with the predicted deletion should make a product. Single cell clones KO3.4 B5 and E1 made a product and wild type or KO5.6 cells did not, as expected.

[0064] FIG. 3 illustrates an example Sanger sequencing of PCR product shows KO3.4 pool, KO3.4 B5, and KO3.4 E1 have the same sequence at the Cas9 cut site.

[0065] FIG. 4 illustrates an example gel shown using primers TP53_exon_4_F and TP53_exon_4_R, wild type exons should make a 496 bp product and TP53 KO5.6 knockouts should make a 344 bp product. KO3.4 B5 and E1 also display a wild type-sized product, indicating these knockouts have a large deletion on one allele and a wild type sized allele.

[0066] FIG. 5 illustrates an example Sanger sequencing of PCR product shows KO5.6 A4, A5, A6, A7, A8, and E3 at the Cas9 cut site. The KO5.6 sequence matches to the predicted deletion, but the sequence becomes rougher after the Cas9 cut site when reading from F or R. This can indicate that the two alleles were cut/repaired differently but the knockout was successful.

[0067] FIG. 6 illustrates an example knockout, KO3.4 E1, which shows an insertion of an A at the Cas9 cut site. The sequence is identical to wild type sequence otherwise. This frameshift causes the resulting protein to be non-functional and not p53.

[0068] FIG. 7 illustrates an example western blot displaying protein in wild type cells, lesser or no protein in KO3.4 B5 and E1, KO5.6 A4, A5, A6, A7, A8, E1, and E3, and lesser or a truncated protein in KO3.4D4.

[0069] FIG. 8 illustrates an example plot displaying relative cell count versus concentration of Nutlin-3a. All knockout clones shown display some resistance to MDM2-inhibitor, Nutlin-3a. All wild type clones shown display some sensitivity. MDM2 binds to and degrades p53. Nutlin-3a competitively inhibits MDM2 and when functional p53 present, the cells undergo cell cycle arrest. Certain knockouts are resistant to nutlin up to 10 uM and wild types are more sensitive. Knockouts do not have a functional p53 protein.

[0070] FIG. 9 illustrates an example plot displaying AUC of TP53 KO pools vs AUC of TP53 wild type pools following 10 day drug treatment. See Table 2 for data points.

TABLE-US-00003 TABLE 2 Approved oncology drug set IV Drugs and their respective areas under the curve (AUC) upon ten-day drug treatment. MCF7 WT TP53 KO TP53 KO Parental pool 3.4 pool 5.6 pool AOD-IV Drug (AUC) (AUC) (AUC) Abiraterone 8.378 8.785 9.254 Afatanib 2.734 2.48 2.139 Alectinib 5.763 4.51 2.7 Allopurinol 8.893 9.837 8.784 Altretamine 8.059 8.874 8.473 Amifostine 8.456 9.108 9.73 Aminolevulinic Acid 8.749 9.101 8.787 Anastrozole 8.01 8.067 8.828 Arsenic Trioxide 5.018 5.242 5.373 Axitinib 2.581 3.743 3.786 Azactidine 8.861 9.05 6.373 Belinostat 1.423 1.525 1.322 Bendamustine Hydrochloride 9.021 9.503 10.23 Bleomycin Sulfate 3.922 4.694 3.421 Bortezomib 0.6974 0.673 0.7486 Bosutinib 8.686 9.254 9.588 Busulfan 9.118 9.336 8.834 Cabazitaxel 1.406 1.658 1.713 Cabozantinib 4.126 4.376 4.52 Capecitabine 9.004 9.208 9.284 Carboplatin 8.83 9.302 8.343 Carfilzomib 0.6359 0.6462 0.6678 Carmustine 9.622 9.826 9.965 Celecoxib 8.779 9.053 9.654 Ceritinib 1.637 1.482 1.421 Chlorambucil 16.08 16.5 17.6 Cisplatin 7.335 6.27 7.44 Cladribine 5.569 3.767 3.956 Clofarabine 5.232 3.085 3.74 Clyclopamine 9.488 9.033 9.093 Cobimetinib 7.621 4.851 8.041 Crizotinib 2.549 2.325 2.941 Cyclophosphamide 16.16 15.2 16.92 Cytarabine Hydrochloride 4.771 2.567 3.381 Dabrafenib Mesylate 14.9 14.73 16.12 Dacarbazine 17.63 16.09 17.2 Dactinomycin 2.01 1.588 1.65 Dasatinib 4.282 4.014 4.43 Daunorubicin Hydrochloride 0.7798 0.8286 0.8964 Decitabine 4.065 4.095 5.295 Dexrazoxane 8.317 8.753 8.868 DMSO 9.021 9.503 10.23 Docetaxel 1.432 1.639 1.663 Doxorubicin Hydrochloride 0.9435 0.9013 1.009 Enzalutamide 8.931 8.864 8.634 Epirubicin Hydrochloride 0.8944 0.9399 1.019 Erismodgib 7.435 6.922 7.491 Erlotinib Hydrochloride 7.115 7.091 7.232 Estramustine Phosphate Sodium 9.864 9.491 9.348 Etoposide 2.93 2.818 3.06 Everolimus 4.699 2.251 3.834 Exemestane 8.564 8.275 8.69 Floxuridine 2.391 1.85 1.997 FludarabinePhosphate 8.551 8.135 7.936 Fluorouracil 6.548 3.782 4.286 Fulvestrant 4.6 3.797 4.145 Gefintinib 8.974 7.676 8.242 Gemcitabine Hydrochloride 2.888 1.464 1.827 Hydroxyurea 9.246 8.069 8.455 Ibrutinib 6.35 5.166 5.711 Idarubicin Hydrochloride 0.958 0.7035 0.8152 Idelalisib 7.566 7.039 7.03 Ifosfamide 9.772 10.26 9.935 Imatinib 8.269 9.674 9.312 Imiquimod 9.496 9.359 9.449 Irinotecan Hydrochloride 3.291 4.015 3.214 Ixabepilone 1.397 1.543 1.473 Ixazomib 0.719 0.7086 0.8766 Laptinib 5.364 5.619 5.857 Lenalidomide 8.724 8.782 8.801 Letrozole 9.288 9.219 9.099 Lomustine 9.62 9.629 9.491 Mechlorethamine Hydrochloride 6.077 6.543 6.171 Megestrol Acetate 9.824 9.221 9.555 Melphalan Hydrochloride 5.766 6.158 5.95 Mercaptopurine 6.103 5.941 5.636 Methotrexate 3.301 4.08 3.1 Methoxsalen 8.476 10.56 10.48 Mitomycin 1.122 1.003 0.912 Mitotane 5.932 11.01 10.17 Mitoxantrone 2.338 0.7284 0.6783 Nelarabine 8.659 11.04 10.08 Nilotinib 5.834 6.967 4.891 Nutlin3 3.686 9.455 8.605 Olaparib 5.48 8.58 7.433 Omacetaxine Mepesuccinate 0.7749 0.7692 0.7717 Osimertinib 4.149 4.056 4.044 Oxaliplatin 2.372 5.272 5.118 Paclitaxel 1.453 1.453 1.432 Palbociclib 3.461 2.923 3.234 Panobinostat 0.6418 0.5862 0.5971 Pazopanib Hydrochloride 6.767 6.259 6.349 Pemetrexed Disodium Salt 2.501 2.438 2.852 Heptahydrate Pentostatin 10.31 9.855 9.32 Pipobroman 8.767 8.997 7.66 Plerixafor 9 8.893 8.524 Plicamycin 0.7344 0.767 0.6852 Pomalidomide 10.08 9.902 10.13 Ponatinib 3.312 2.944 3.632 Pralatrexate 2.822 2.774 2.86 Procarbazine Hydrochloride 8.119 10.04 10.7 Raloxifene 5.361 6.017 6.562 Regorafenib 3.995 5.541 5.25 Romidepsin 0.5862 0.6551 0.5264 SenexinB 5.203 6.869 6.954 Sirolimus 2.534 2.623 3.106 Sorafenib 3.981 5.27 4.98 Streptozocin 9.145 10.58 9.912 Sunitinib 3.556 3.076 3.758 TamoxifenCitrate 7.863 5.949 7.798 Temozolomide 9.187 9.565 10.06 Temsirolimus 2.486 1.939 2.767 Teniposide 1.342 0.9417 1.041 Thalidomide 7.13 9.18 10.21 Thioguanine 4.122 4.936 4.789 Thiotepa 3.965 5.443 5.535 Topotecan Hydrochloride 0.7919 0.7814 0.8044 Trametinib 7.726 6.641 8.533 Tretinoin 7.394 7.809 7.997 Triethyleneme1amine 2.223 2.224 2.319 Trifluridine 2.698 2.372 2.626 UracilMustard 9.331 9.337 9.516 Valrubicin 1.194 1.25 1.05 Vandetanib 2.871 2.831 3.022 Vemurafenib 8.722 7.987 8.365 Vinblastine Sulfate 1.393 1.54 1.382 Vincristine Sulfate 1.668 1.776 1.54 Vinorelbine Tartrate 1.616 1.797 1.629 Vismodegib 8.801 8.017 8.363 Vorinostat 2.073 2.202 2.114 Zoledronic Acid 9.222 9.268 9.417

[0071] FIGS. 10A-10D illustrate sensitivities of MCF7 wild type and Knockout cell lines to Nutlin-3a, Fluorouracil, Oxaliplatin, and Palbociclib.

[0072] FIGS. 11A-11C illustrate that Nutlin resistance can be predictive of drug responses in oxaliplatin, fluorouracil (5FU), and palbociclib (Palb). Adjusted R-square values are shown. Values plotted are the areas under the curve for each treatment. See FIG. 8 and Table 3.

TABLE-US-00004 TABLE 3 Select anticancer drugs from the AOD-IV and their respective areas under the curve (AUC) upon ten day drug treatment on single cell clones. Cell line 5-Fluorouracil Nutlin-3a Oxaliplatin Palbociclib PR WT Pool 2.156 1.16 0.9557 2.446 PR WT A8 2.126 0.9589 0.7779 2.127 PR WT B8 1.886 0.8828 0.7911 1.917 PR WT C7 2.105 1.042 0.8311 2.543 PR WT G2 2.265 1.011 0.8186 2.542 KO 34 Pool 1.721 1.452 1.25 1.88 KO 34 B5 1.618 1.234 0.973 1.522 KO 34 D4 1.812 1.354 1.392 1.826 KO 34 E1 1.945 1.538 1.46 2.274 KO 56 Pool 1.745 1.374 1.164 1.457 KO 56 A4 1.716 1.568 1.29 1.846 KO 56 A5 1.46 1.595 1.197 1.647 KO 56 A6 1.811 1.512 1.243 1.681 KO 56 A7 1.81 1.761 1.23 1.753 KO 56 A8 2.02 1.656 1.229 2.031 KO 56 C5 1.826 1.569 1.239 1.632 KO 56 E1 1.697 1.492 1.208 1.497 KO 56 E3 1.645 1.564 0.9635 1.469

[0073] While certain embodiments of the disclosed subject matter have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the subject matter.

[0074] These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood the aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the invention further described in the appended claims.