CORE C- FA CELL RESPOSITORY CORE

CORE C-FA 细胞储存库核心

基本信息

批准号：
7650189
负责人：
Markus Grompe
金额：
$ 15.9万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7650189
关键词：
3p25.3 Academic Medical Centers Accidents Accounting Affect Alabama Alkylating Agents Alleles American Anemia Antibiotics Antigens Apoptosis Appendix Applications Grants Aspirate substance BRCA2 gene Baltimore Berlin Biochemical Biological Assay Biology Biopsy Blood Bos taurus Boston California Canada Cancer Center Carbon Dioxide Catalytic Domain Categories Cattle Cell Cycle Cell Line Cell fusion Cell hybridization Cells Cellular biology Child Chromosome Breakage Chromosome Transfer Chromosomes Cities Clinical Clinical Sciences Collaborations Colorado Communication Complement Complementary DNA Computers Consent Forms Cultured Tumor Cells DNA DNA Damage DNA Interstrand Crosslinking DNA Repair DNA biosynthesis DNA crosslink Dana-Farber Cancer Institute Databases Defect Denmark Detection Digestion Disadvantaged Disease District of Columbia Documentation Dose Double Strand Break Repair Ear Eligibility Determination Embryo Ensure Environment Ethanol European Excision Experimental Hematology FANCD2 protein FANCG gene Facility Construction Funding Category Failure Family Family Planning Family member Fanconi anemia protein Fanconi&apos s Anemia Fibroblasts Ficusin First Degree Relative Foundations Foxes France Fred Hutchinson Cancer Research Center Freezing Funding Future G Cells Generations Genes Genetic Genetic Complementation Test Genome Genomics Genotype Germany Glutamine Goals Growth Harvest Health Sciences Hematology Hospitals Hour Human Human Genetics Human Herpesvirus 4 Hybrid Cells Hybrids Ice Immunogenetics Incidence Incubators Individual Infection Information Storage Informed Consent Institutes International Internet Iris Italy Japan Joints Jordan Journals Kidney Kinetics Knockout Mice Label Laboratories Large T Antigen Leadership Left Liberase Link Liquid substance London Mails Malignant Neoplasms Mediating Medical Medical Genetics Medical Oncology Medical Records Medical center Medicine Metabolism Methods Michigan Minnesota Mitomycin Molecular Molecular Genetics Molecular and Cellular Biology Mus Mutant Strains Mice Mutation Mutation Analysis Neomycin Netherlands New Jersey New Mexico New York Newly Diagnosed Newsletter Nitrogen Nuclear Numbers Oncogenes Ontario Oxygen Paper Parents Paris, France Pathology Pathway interactions Patients Pediatric Hospitals Pediatric Oncology Pediatrics Pennsylvania Pharmacologic Substance Phase Phenotype Philadelphia Polymerase Chain Reaction Population Population Group Prenatal Diagnosis Principal Investigator Procedures Process Program Research Project Grants Protein Isoforms Protein Overexpression Protocols documentation Psoralens Public Health Publications Puromycin Quality Control Quebec Questionnaires RNA Radiation Radiation Oncology Range Rate Reaction Reagent Registries Regulation Reporting Research Research Personnel Resistance Resources Rest Retroviral Vector Retroviridae Rodent SV40 T Antigens Sampling San Francisco Schools Science Serum Serum-Free Culture Media Shipping Ships Siblings Signal Transduction Simian virus 40 Skin Solutions Somatic Cell Genetics Source Spain Speed Standards of Weights and Measures Stem cells Surgical Flaps Techniques Telephone Telomerase Testing Texas Therapeutic Radiology specialty Time Tissues Toxicology Transfection Tube United States United States National Institutes of Health Universities Vesnarinone Virginia Washington Week Western Blotting Woman Work Workload antibiotic G 418 anticancer research attenuation base carcinogenesis cell bank cell growth cell transformation cellular transduction clastogen clinical Diagnosis collagenase college day demographics density dispase epstein barr virus mediated immortalization established cell line expression vector falls fetal flasks gene therapy genetic pedigree grandparent hygromycin A immortalized cell indexing interest kidney epithelial cell lymphoblast medical schools moran A mutant novel positional cloning programs rapid growth rapid technique repository research study response restoration sample collection seal stem success tissue culture tool

项目摘要

from 1998-2003 Since its inception in 1992, the repository has taken in 635 patient samples. 403 cell lines have been established, both from FA patients, FA family members and patients with disorders similar to FA (34 samples)_ In total we have 271 established cell lines from FA patients. Of these 100 are EBV transformed lymphoblasts, 160 are primary fibroblasts and 11 are immortalized fibroblasts. We have 3 liquid nitrogen tanks and the samples are divided between the 3 in order to protect ourselves against a freezer accident. At the time the sample(s) is sent to us each family is requested to sign a consent form and fill out a detailed clinical questionnaire (sample questionnaire is attached). Using funds from the program project grant, we hired a computer programmer to create a tailor-made database which links family demographics, clinical information, mutation analysis and complementation group results and sample storage data, including the liquid nitrogen tanks. This database allows us to rapidly compile information and keep track of stored samples. 267 Principal Investigator: Moses, Robb E. IA. Cell line recipients Cell lines generated by Core C have been used distributed to labs around the world. Over 1500 worldwide. Shipments average 10 cell lines/per investigators which have requested and received the table below. by PPG investigators in all 3 projects and widely cell lines have been shipped to investigators request and 2-3 requests/investigator. A list of samples from Core C from 1994-2003 is given in Rajni Agarwal Childrens Hospital Medical Center [..Cincinatti, OH Arleen Auerbach Rockefeller University New York, NY Juan Beuren CIEMAT : Madrid, SPAIN Alan B_el/Vivian Cheung University of Pennsylvania Chidlren's Hospital Philadelphia, PA Colin Campbell University of Minnesota Minneaplolis, MN Martin Digweed Institute for Human Genetics Humboldt University of Berlin Berlin,: GERMANY Jean Paul Feugas Hopital St Louis Paris: France Konstantin Galaktionov Bayl0r College of Medicine Houston; TX: Paul Harris _ Molecular and: Ceil Biology Univl of California, Davis CA George Washington University Washington, DC Thanos Halazonitis Wistar Institute U Penn Philadelphia, PA Hans Joenje Institute of Human Genetics Free University, Amsterdam THE NETHERLANDS Steven Arkin The Mount Sinai Medical Center, New York, NY Abida Awan Immunogenetics Laboratory SL Mary's Hospital Manchester; U.K. Bill: Brinkley Baylor College of Medicine Houston, Texas Madeleine Carreau University Laval Quebec City, Quebec CANADA Paolo Degan IST Genoa, ITALY Harry Drabkin Medical Oncology University of Colorado Health Sciences Cemer, Denver, CO Bernard Fox OHSU Portland, OR Thomas Glover University Of Michigan Ann: Arbor, Mt Mike Heinrieh OHSU Portland_ OR ' Merle Hoekstra Signal Pharmaceuticals San Diego, CA Nigei Jones Donnan Labs University of Liverpool Liverpool, UK Christan Kuehne ICGEB Trieste, ITALY 268 Raghbir S. Athwal Temple University Philadelphia, PA Manuel Buchwald Hospital for Sick Children Toronto, Ontario Canada' = ' Stere Brodie University of New Mexico Albuqueque, NM Pia Marie Cosmos TIGEM Napoli, ITALY Alan D'Andrea Pediatric Oncology Dana-Farber Cancer Institute Boston, MA William D Fergusson Cell bank, Immunogenetics laboratory. St. Mary's hospital Manchester, UK Franceseo Galimi The Salk institute LaJolla, CA Phiippe Guardiola Fred Hutchinson CRC Seattle, WA Maureen It0atlin OHSU Pomand;OR ,,,. Frank Jirik Hsc : Calgary, AB CANADA Aimee aae'kson University of Washington Seattle, WA Albert Kheradpour The Univ. of Texas Med. Branch at Galveston, Children's Hospital Galveston, TX Gary Kupfer University of Virginia Charlottesville, VA Janet Lewis Clinical Sciences Center Madison, WI Lei Li Dept. Radiation Oncology M.D, Anderson Cancer Center Houston; TX Marcello Merola Instituto di Chimica Biologica Univetsita degli studi di Verona, ITALY Peter McItngh Cancer Research UK Labs University of Oxford Oxford.,... UK Ryuichi Okayasu International Space Radiation Lab Chiba, JAPAN Janice Pluth Lawrence Livermore Labs Berkeley, CA Fayruz Rassool Rayne Institute Guy's, Kings & St. Thomas's School of Medicine London, UK Jann Sarkaria Mayo Foundation Rochester , MN Olivia Smith Department of Pathology Bay!or College of Medicine Mt. Sinai School of Medicine NY, NY Tim Townes Dept. ofBiochem. & Mol. Genetics, Univ. of Alabama at Birmingham Birmingham, Alabama Mike Whitney . San Diego, CA BonnieKing Dept. of Therapeutic Radiology Yale University School of Medicine New Haven, CT Johnston Liu Hematology Branch, NHLBI Bethesda, MD Ray Monnat Jr. Dept. of Pathology University of Washington Seattle, WA Stephen M. Meyn Yale University School of Med. Dept. of Human Genetics New HaVen, CT .... Chris Mathew Guy"s Hospital London, UK : Randall:Phelps Fred Hutchinson Cancer Research Center Seattle, WA Sharon E. Plon Dept. of Pediatrics Baylor College of Medicine Houston, TX Filippo Rosselli Institute Andre Lwoff Villejuif, FRANCE Leona D. Samson Dept. of Molecular and Cellular Toxicology Harvard University Public Health Boston, MA Maulik Shah St. Louis U: Cancer Ctr St. Louise MO BIDMC Harvard Medical School Boston, MA Jean-Michel Vos UNC Lineberger Cancer Center University of NC at Chapel Hill, Chapel Hill, NC Johan de Winter Free University Amsterdam, THE NETHERLANDS 269 PrincipalInvestigator:Moses,RobbE Muriel W. Lambert : Department of Pathology UMDNJ - New Jersey Medical School Newark, NJ Uma Lakshmipathy Stem Cell institute University of Minnesota Minneapolis, MN Junia V. Meio Dept. of Hematology-ICSTM Hammersmith Hospital London U.K. Sankar Mitra UTMB Galveston, TX Institute for Cancer Studies University of Sheffield Sheffield, UK K J Patel MRC Laboraotory of Molecular Biology Cambridge, UK " Tony Parks Dept. of Pathology University of Washington Seattle, WA Jordi Surralles University of Barcelona , Barcelona, SPAIN ¿ Dennis Simpson Hematology Branch, NHLBI Bethesda, MD Iris Schrijver Stanford University Medical Center Stanford, CA Departnmnt of Medical Genetics Glostrup - DENMARK Weidong Wang Lab of Genetics NIH Baltimore, MD Matthias Wahl UCSF San Francisco, CA Principal Investigator: Moses, Robb E. ..C...hris Walsh Hagop Youssoufian Margaret Zdzienicka [ Mt. Sinai School of Medicine Brigham and Woman's Hospital Leiden Univeristy NY, NY Boston, MA ATH1ELeNidEeTnH, ERLANDS lB. Repository Publications Several papers have resulted from use of repository cell lines in the last funding cycle (1999- 2003). In addition, the repository has generated several independent publications. Papers involving the use of repository resources are listed below. P. M. Jakobs,:L: Smith, M. Thayer and M. Grompe (t999)"Construction ofMonochromosomal Hybrid Cell Lines containing Single Murine Chromosomes using Embryonic Stem Cellswith Targeted Mutations asrnicrocell donors", M_alian Genome 10:381-4 : M. N: Akkafi, K Bateman, C. A, Reifstec_ S. B: Oison and M, Grompe (2000) "DNA replication is required to elicit cellular responses to psoralen induced interstrand DNA crosslinks", Molecular and Cellular Biology 21' 8283-9 J..A. Hejna,, C.D. Timmers, C. Reifsteck, DA. Bruun, L.W. Lucas, P.M. Jakobs, S. Toth-Fejet, N. Unsworth, S.L. Clemens, D.K. Garcia, S.L. Naylor, M.J. Thayer, S.B. Olson, M. Grompe and R.E Moses (2000) "Localization of the Fanconi Anemia Complementation Group D Gene to a 200-kb Region on Chromosome 3p25.3" American Journal Human Genetics 66, 1540-1551 C. Timmers, T. Taniguchi, J. Hejna, C. Reifsteck, L.Lucas, D. Bruun, M. Thayer, B. Cox, S. Olson, A.D. D'Andrea, R. Moses and M. C_ompe (2001) Positional cloning of a novel Fanconi Anemia gene, FANCD2. Molecular Cell 7:241-246. K. Nakanishi, A Moran, T. Hays, Y. Kuang, E. Fox, D. Garneau, R.M. de Oca, M. Grompe, A.D. D'Andrea AD (2001) "Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9", Experimental Hematology 29 (7): 842:9 Y. M. N. Akkari, R. Bateman, C A, Reifsteck, A.D. D'Andrea, S, B. Olson and M. Grompe (2001) "The 4N cell cycle delay in Fanconi anemia reflects growth arrest in late S-phase", Molecular Genetics and Metabolism (74): 403-12 N. G. Howlett, T. Taniguchi, S. Olson, B. Cox, Q Waisfisz, C. De Die-Smulders, N. Persky, M. Grompe, H. Joenje, Pals G, H. Ikeda, E.A. Fox and A.D. D'Andrea (2002) "Biallelic Inactivation of BRCA2 in Fanconi Anemia" Science 297 (5581): 606-9 M. Digweed, I. Demuth, S. Rothe, R. Scholz, A. Jordan, C. Grotzinger, D. Schindler, M. Grompe and K Sperling "SV40 large T-antigen disturbs the formation of nuclear DNA- repair loci containing MRE11" Oncogene 21(32):4873-8 270 PrincipalInvestigator:Moses,RobbE. M. Digweed, S.Rothe,I. Demuth,R. Schotz,D. Schindler,M. Stumm,M, Grompe, A. Jordan and K, Sperling (2002)"Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia" Carcinogenesis 23(7): 1121-1 t26 A. Folias, M. Matkovic, D. Bruun, S. Reid, J. Hejna, M. Grompe, A.D. D'Andrea and R. Moses. (2002)"BRCA1 interacts directly with the Fanconi anemia protein FANCA', Human Molecular Genetics 11 (21):2591-7 A. Rothfuss and M. Grompe (2003) "The kinetics ofinterstrand human cells: Implications for the Fanconi anemia_RCA1 Cellular Biolog% in revision R: Lundberg, M Mavinakere and C. Campbell (200t) "Deficient .... extracts:from Fanconi anemia fibroblasts'; JBC 276 (12): S. L. .D..o..n..a..h..u..e. and C. Campbell: (2002) "A D NA :doub 1 e strand anemia fibroblasts", Ji3C 277(48): 46243-46247. S. L. Donahue, R. Lundberg, R. Saplis and C. Campbell (2003) double-strand break repair in Fanconi anemia fibroblasts", 1C. Collaboration with other repositories DNA crosslink removal in pathway", Molecular and : :: DNA end joining activity in 9_43.95491 bleak repa!_ defect in Fanconi "Deficient regulation of DNA JBC 278 (32): 29487-29495. Two other repositories for FA patient samples exist. Here in the United States, the International Fanconi Anemia Registry (NAP,) has been in existence for 10 years under the leadership of Dr. Arleen Auerbach at Rockefeller University, New York. However, access to those samples has been limited for outside investigators and this has created the need for a repository which will ....... ¿ H distribute material w_th, "no stnngs attach.... ed In Euro P e, the: group :of : Ha: ns Joe: nje has established a repository: There has beena division Of labor between the European Repository and the OHSU repository _nd we have a collegial working relationship. There are several joint pubhcatlons (1-4)Joenje s group ocuses on lymphoblast lines; whereas we have emphasized the establishment offibroblast lines, especially i_o_alized fibroblast _esl Each Cell t_e has its unique advantages and disadvantages: Lymphobtastsare easy to establish and serve as agood Source of _A andDN for mutation analysisl However; _they are transformed andtherefore less suitable for studies of ce!t CyCle COntrol and apoptosis N_ primary cellsi: In addition, they are not good ieCipientS for miCrOcell:mediated chtomosoNe transfer and havea high rate of somatic reversion (5). Primary fibroblasts are excellent tools for the study of primary defect in FA, because they are not transformed and have not acquired secondary phenotypes related to rapid growth and transformation. Immortalized fibroblasts are the cell line of choice for microcell- mediated chromosome transfer and for the establishment of clonal transfectants. 2. Functions of Core C for the current application Because of goals already achieved and changes in the field, the priorities of Core C will be different from the previous grant application, As we have already collected samples from many of the known FA families in the United States, we will re-focus our efforts on determining the 271 PrincipalInvestigator:MosesiRobbE. complementationgroupsof allpatientsalreadyin therepositoryandontheestablishmenotf immortalizedfibrobtastlinesfrom FA complementatiognroupsfor whichthegenehasnot yet beencloned.We will alsocontinueto acceptsamplesfrom newpatients,establishcelllinesand performcomplementationgroupassignmenatsdescribedbelow. In addition,we will generateimmortalcelllinesfrom murineFA modelsandperformshortterm cultureof tumorsderivedfrom FA mice.Both murineandhumancellsfrom defined complementationgroupsareof importancefor projects 1, 2 and 3. 2A. Establishment of cell lines, storage of DNA and RNA Identification of families We plan to contact newly diagnosed families and patients with the help of the Fanconi Anemia Research: Fundl The majority of North _erican families with FA are in contaCt with the Fund. Since the Fund regards the establishment of a cell repository as a need, they have been willing to help establish: contact with families. In the FA newsletter or during other routine communications, families are informed: about our attempts to obtain samples and are then encouraged to contact us by phone _rangements for obtaining and mailing the samples are then made with the family or their physiciani We are interested cells from a) individual patients; b) multiplex families' and c) singleton families. We plan to obtain samples from patients and analyze them by retroviral complementation (see below). If the patient has an unknown complementation group or has an unusual clinical presentation, we will also obtain samples from all first degree relatives, i.e., parents and siblings, as well as grandparents when available. For financial and time reasons no attempt will be made to immortalize these cells. However, at least 200 gg of genomic DNA will be stored from each individual (-20 ml blood), enough for 2000 genotyping experiments using PCR based markers. Sample collection and processing Samples will be collected and processed as described in the following: In all individuals affected with the disease, regardless of whether they are part of a multiplex- or consanguineous family two 10 ml tubes of blood will be drawn and a skin biopsy performed to derive primary fibroblasts, One tube of blood will be used to isolate genomic DNA by a simple salting out procedure (6) The second tube will be utilized for Epstein-Barr virus (EBV) mediated immortalization of B-Iymphocytes (7, 8). Lymphoblasts Several workers in the field have reported great difficulty at obtaining transformed FA lymphoblast cell lines, with failure rates routinely as high as 30-50%. However, improvements in technique utilized by us have reduced the incidence of this problem. An important technical detail in the establishment of FA cell lines is to use a 1% oxygen environment to enhance cell growth. This is achieved by gassing the tissue culture flasks with a mix of 95% N2 and 5% CO2 and then tightly sealing them. The cells are also gassed at each media change. In order to ensure transformation, we also routinely expose cells to a second dose of EBV, if transformation isn't obvious by 10 days, After establishment of a cell line, RNA (9) (50 lag) and DNA (6) (200 lag) will be isolated by standard methods prior to freezing them down. This will serve as a backup for linkage and mutation studies in case of culture failure upon thawing. 272 PrincipalInvestigator:Moses,RobbE. Fibroblasts Primary skin fibroblastscultureswill be establishedfrom skin biopsiesof patientswilling to participatein the study.For someresearchapplicationsprimarycellsoffer someadvantagesover transformedlymphoblastsandarethereforeimportantto establishin at leastsomefamilies(see below). Recordsand documentation All families will be asked to sign an informed consent for the sample donation, In addition, they will be asked to fill out a questionnaire regarding the pedigree and clinical information about the presentation of the disease (see appendix). Summaries of medical records will be obtained, especially regarding the results of chromosome breakage studies, which we regard as the only objective criteria for FA_ False clinical diagnoses are a serious source of possible error in any linkage or complementation Study. The result of a chromosome breakage study after clastogen exposure is required for all FA patients and all siblings. These results are especially important for siblings, since FA patients can be entirely asymptomatic for many years, whereas the biochemical abnormalities are present even prenatally. The cells of any patient or sibling without well documented chromosome breakage studies, will be analyzed after mitomycin C (MMC) challenge 2B. Complementation group analysis by retroviral correction Studies performed by cell-cell fusion experiments and mutation analysis have shown that FANCA mutations account for -65%, FANCG mutations 15% and FANCC mutations for 10% of all FA cases (10-13). All other known complementation groups together represent only 10 % indicating that they are much more rare in most populations than groups A, G and C. To date 11 distinct complementation groups have been proven to exist (A, B, C, D1, D2, E, F, G, I, J, L) but it is possible that the number ofFA complementation groups is even higher (10,22). There are 2 strong reasons to determine the complementation group status of all cell lines in our repository: First, the patient families want this information, because it allows accurate, DNA based prenatal diagnosis and carrier detection and because it establishes eligibility for gene therapy trials. Second. it is important to identify all FANCA, C, D2, 13, F, and G cell lines in order to focus our efforts offibroblast immortalization on cells from yet-to-be cloned or rare FA complementation groups. Only I0%-15%: &cells fall into this category and thus our work load can be greatlY reduced if we can identify these cells of interest. Because very few mutant alleles account for the majority of patients, mutation analysis can detect >95% of FANCC patients (14, ! 5): However, mutations in FANCA and FANCG are much more with Alan D' Andrea's group at Harvard) have developed a more simple and rapid method for complementation group assignment in patient fibroblasts (1, 21, 23). The method relies on retroviral correction of the MMC sensitivity of patient cells and retroviral restoration of the DNA- damage-inducible monoubquitination of the FANCD2 protein, described below. Primary fibroblasts are plated at low density into 6 well plates. One or two days after plating, media is replaced with 3/4 serum free media containing 8 tag/ml ofpolybrene and 1/4 retroviral supernatant, either FANCA, C, D2, E, F, or G. Plates are then returned to the incubator. After two to four hours an equal volume of complete media (alphaMEM supplemented with 15% fetal bovine serum, L-Glutamine and antibiotics) is added and cells are returned to the incubator. The 273 = i : : Principal Investigator: Moses, Robb E. : : : next morning the retrovirat containing media is replaced.with complete media and 48 hours after infection; cells are put under puromycin selection (1 gg/ml)_ Non infected cells serve as controls. Cells are grown to confluency and then expanded into three 150 mm dishes, one for freezing, two for FANCD2 westerns (see below) and one T25 tissue culture flask which is sent to Core: B for chromosome breakage testing and complementation group assignment by retroviral correction of MMC sensitivity. The described:protocol is easy and the generation of retroviratly transduced cell populations is rapid. Chromosome breakage analysis by Core B is the most time consuming step. To alleviate some of the burden placed on Core B, and to speed up the process, FANCD2 western blots, will also be used to determine complementation groups (see below). Thus far, 92 cell fines definitively assigned : FANCA: 48 F_CC: 8 :' FANCD2:2 : F_CEi 1 have been retrovirally infected and 63 primary fibroblasts have been to complementation groups based on retroviral complementation: : : : , : 1 FANCG: 2 29 studies are incomplete, 10: out of range, 8: non A,C,D2,F 1" non A,C 3" need to re-do 7: pending 62 cell lines have not yet inconclusive or pending: normal or mosaic or G been retrovirally infected. We also plan to generate unassigned celt lines (22, a FANCL retrovirus construct which will be used to screen the 1). FANCD2 Western Blots : To dete_ne whether or not unassigned cell lines can express the: active, monoubiquitinated isoform ofF_CD2 _ANCD2L), FANCD2 western blots will be done (20. 2!)i _ mutations in any ofthe FApmteins upstre_ from FANCD2: (F_CA' :Bi C; E, F, G. L)results in the loSs of DNA2damage:inducible FANCD2 mon0ubiquitination, only F_CD2S (the shorter: :: : D2, E, F, G, or L, e.g., FANCD 1/BRCA2. FANCD2 FANCD2S (see flow chart below). Therefore, complementation group assignments can well as by chromosome breakage analysis (Core B). wilt be transduced with retroviral vectors containing undergo both FANCD2 western blot and chromosome deficient cell lines will have no or truncated be made by FANCD2 western analysis as Cell lines that express only the FANCD2S FANCA, C, D2, E, F, G, L cDNAs and then breakage analysis (see flow chart below). Additionally, FANCD2 western blots are/will be used for 'quality control' testing of harvests of FANCA, C, D2, E, F, and G retroviral supernatants. 274 PrincipalInvestigator:Moses,RobbE 2C. Generation of immortalized fibroblasts from non-A, non-C, non-D, non-E, non-F, non- G and non-L FA patients Immortalized fibroblast cell lines are an invaluable reagent for many cell biology and somatic cell genetic experiments. We have succeeded in creating a number of new, immortal FA lines (10) and two of these, PD-20 and ELTA423, have been key to identifying and locating the FANCD1/BRCA2 and FANCD2 genes (18, 19). In the past, we used randomly selected cell lines for our efforts. However, because of the high representation of complementation group A, most of our new celt lines were FANCA. Cell-cell fusions (11) and the retroviral complementation assay (1) described above have allowed us to identify several cell lines from undefined complementation groups which can be subjected to our immortalization protocol. So far, we have 8 cell lines from yet undefined complementation groups. We have immortalized three of these and the rest are in the process of being immortalized (2). Fibroblast immortalization protocol Previously our immortalization protocol was as followS: Primary fibroblasts were electroporated with pSV7, an expression: construct for SV40 small t-antigen. This routinely leads to transformation and rapid growth within 2-4 weeks. The SV40 transformed cells were then mutagenized with ethylmethylsulfonate (EMS), an alkylating agent, to accelerate growth promoting mutations. These cells were then serially passaged until they either underwent crisis or became immortal (> 100 cell doublings). Our success at generating immortal lines in this setting was - 1/3. The protocol has now been modified as follows: After SV40 transformation, it is no longer necessary to mutagenize the cells with EMS. Instead, cells are electroporated with an expression construct for the catalytic subunit of human telomerase (hTERT; 17). This reagent has been made available to us from Geron Corporation. While telomerase expression alone does not produce an immortal phenotype, it is a required step and the chance of immortalization is greatly increased. We have subcloned hTERT into several expression vectors which contain different setectable markers, including neomycin-resistance, hygromycin resistance and puromycin resistance. Cells are electroporated separately with hTERT constructs containing either the G418 resistance or hygromycin resistance marker and exposed to selection :media to produce telomerase expressing clones. These are then be serially passaged until > 100 population doublings have been achieved: Ten celt lines have been immortalized in this fashion. Complementation group assignment New immortal FA fibroblast lines might belong to the same or distinct complementation groups. Therefore, cell lines will be transfected with BRCA2 cDNA and also grouped by standard cell-cell fusion technique with FANCB, FANCD 1/BRCA2 cell lines until retroviral vectors from newly cloned complementation group become available. If these experiments indicate that a cell line does not belong to any of these complementation groups, we will generate whole cell hybrids between the new cell lines themselves. In these fusions, the parental cell lines will have been marked with different dominant selectable markers. Typically all newly immortalized cell lines will already be puromycin resistant (selection for the telomerase expression construct). Therefore, we will use the hygromycin resistance and neomycin resistance markers to introduce a separate markers for cell fusions. Double selection in both hygromycin and neomycin will yield whole cell 275 PrincipalInvestigator_Moses,RobbE. hybrids,whichwill beanalyzedby Core B for cytogeneticorrectionandWesternblot analysisif thenewcell lineis 'upstream' from FANCD2 (see flow chart below). For each new FA cell line, we will seek to determine to which of the complementation groups defined by Hans Joenje (10, 22) the new line belongs. Unfortunately his cOmplementation test is based on lymphoblast lines, whereas we use fibroblasts. In those cases where we have a lymphoblast line from the patient, we will mail the cells to Dr. Joenje to be fused with his reference cell lines. If we don't have a lymphoblast line for fusions, the new cell line will be assigned a temporary designation and we will contact the family in order to obtain samples. We have been informed by Dr. Joenje that he is obtaining fibroblast lines from all his "index" patients. We therefore expect to be near future. : : : I A, l I breackhaCrogOemRoEsaonmaBelysis I I J comptementat on I arouo assignment II I (o_of'MMC sensitiv t" _ I"1 i . .s H I....... _ '1 : I':" no complementation I group assignment I on:i_ I ! =::for !_ I whole ceil fusions ! IBRCA2 cDNA transfection able to correlate lymphoblast and fibroblast results within the J Cgro¿mupleamsesnigtantmi0ent : :i ! ' : I unassigned ofibr0b!asts : I I FANCD2 western :N0ts i:: i ! .... I FANCD2L FANC(D')2L I (+) I infect with immortalize C, D2, E, F, G, k with retrovirus SV40 T antigen hTERT I I whole cell fusion studies blots transfect BRCA2 cDNA FANCD2 western 1 ! I complementation i Ino complementation group I group assignment I I group assignment I FANCI_2I : :i I ': FANCD2L: : '1 (*) : II (') :_: t:: : : / : _ =: 2D. Establishment Several strains of contrast to human reason, immortal of immortal cell lines from Fanconi anemia mutant mice mice with targeted deletions in FA genes are available in the program project. In cells, rodent cells have a high rate of spontaneous immortalization. For this lines can be obtained without the use of SV40 T-antigen or telomerase RT 276 PrincipalInvestigator:Moses,RobbE_ (TERT) overexpressionS: uchcelllinesarethereforehighlyusefulfor functionalstudieswhich requiretheisolationandstudyofclonal isolates.AdherentlygrowingCellinescanbederivednot onlyfrom fibroblasts;butals0othertissuetypes;particularlyepithelialtissues,suchasthe kidney. To dateWehavegeneratedspontaneouSilmy mortalizedceltfinesfrom theearsandrenal: epitheliumof Fancc knockout mice (one each) and from the ears ofwildtype and Fancd2 mutant mice. We plan to generate similar lines from Fanca, Fancd2 and Blm mutant mice. In addition, lines will be generated from Fancd2/Blm double mutants (project 1) and Fancd2/telomerase deficient mice (project 2). For each immortal cell line, a retrovirally complemented counterpart will be generated. Mouse ear fibroblasts and kidney epithelial cells are generated as follows: Anesthetized and/or euthanized mice are thoroughly wiped down with ethanol. Kidneys and ear flaps are removed mad plaCed in separately labeled TC dishes with cold PBS. In a TC hood using aseptic technique, tissues are Chopped as small as possible (about i mm)and are then digested at 3:7 C in tissue digestion flasks on a stir plate set at 4. Kidneys are digested for 45 min in 3 ml of 0.07 mg/ml Liberase Blendzyme 3 (Roche) in50/50 PBS/DMEMi : : _ : Ear flaps are digested for one hour in3mt of4mg/ml CollagenaSe/dispase (Roche) in 50/50 PBS/DMEM. Kidney and ear tissue then undergo a second digestion. At least 2 ml of supematant is removed from the kidney digest (leaving undigested tissue and clumps) and to stop the reaction is put on ice into a 15 ml conical tube containing 5 ml of DMEM supplememedwith 15% fetal bovine serum (FBS; Gemini) Three more 3ml of Liberase solution (above) is added to the remaining kidney tissues and digested at 37C for another 45 min. For ears, no supernatant is removed. Instead, 3 ml of0.5mg/ml collagenase (Sigma) in 50/50 PBS_MEM is added to the flask and digested for 40-50 min at 37C. After digestion, the kidney digest solution is triturated and then transferred to the conical tube containing the supernatant from the first digest and kept on ice. Similarly, the ear digest solution is also triturated and transferred to a 15 ml conical tube containing 5 ml of DMEM supplemented with 15% FBS and is kept on ice.: Kidney and ear cell digests are then spun at 1000 rpmfor 5 min: Supernatant is aspirated and cells are re.suspended in 3 ml DMEM (supplemented with 15% FBS, 2 mM L-Glut_ne and antibiotics), transferred to 60 TC mm plates and placed in the incubator. used for the establishment of kidney epithelial cells (above) will also be used for the establishment of tumor cell cultures. 2F. To develop a Fanconi anemia web