http://www.refaelilab.com/index.php en pvr@xigent.com Copyright 2009 2009-10-09T18:01:34+00:00 http://www.refaelilab.com/index.php/site/genetics_core/ http://www.refaelilab.com/index.php/site/genetics_core/#When:18:01:34Z Our Aim: The aim of this core is to provide a comprehensive and technically advanced resource for the design, generation and production of lentiviralbased shRNA vectors as well as retroviral approaches to manipulate gene expression in primary cells and cell lines of interest. The services provided by this core will include a number of lentiviral vectors and maps, as well as assistance in the identification of candidate shRNA targeting sequences, cloning into viral vectors and validation in the relevant cellular context, by sequencing and assessment of the efficacy at the protein level. This core will also provide training on the proper compliance with biosafety issues required for lentivirus-based research. We will focus our efforts on generating shRNA encoding lentiviruses aimed at knocking down expression of genes that are of specific importance to the participants of this program. shRNA Validation Protocol: Search target gene for predicted optimal shRNA sequences. Clone primers into shRNA expression plasmid Validate knock down of target gene in DO.11.10 T cells Core Status as of 5.13.05 Gene Relevant Status Bim Apoptosis/Survival Validated Puma Apoptosis/Survival Validated Bax Apoptosis/Survival Cloned Bak Apoptosis/Survival Cloned Bcl-2 Apoptosis/Survival Analyzed Lyn BCR Signaling Validated Syk BCR/TCR Signaling Validated CD22 BCR Signaling Analyzed CD72 BCR Signaling Analyzed PKCg BCR/TCR Signaling Cloned PKCd BCR/TCR Signaling Cloned SHIP BCR/TCR Signaling Analyzed SHP1 BCR/TCR Signaling Analyzed AKT-1 Apoptosis/Survival Analyzed AKT-2 Apoptosis/Survival Analyzed AKT-3 Apoptosis/Survival Analyzed PTEN Apoptosis/Survival Analyzed PDK-1 Apoptosis/Survival Analyzed PDK-2 Apoptosis/Survival Analyzed mTOR/FRAP Apoptosis/Survival Analyzed Foxo-1 Apoptosis/Survival Analyzed Foxo-3 Apoptosis/Survival Analyzed Foxp-1 Apoptosis/Survival Analyzed Foxp-4 Apoptosis/Survival Analyzed ZAP70 TCR Signaling Analyzed LAT TCR Signaling Validated CD8a TCR Signaling Validated c-rel NF-kB Signaling Validated Rel-a NF-kB Signaling Validated Rel-b NF-kB Signaling Cloned iKK-Bi NF-kB Signaling Cloned IkBe NF-kB Signaling Validated IkBa NF-kB Signaling Validated Bcl-10 NF-kB Signaling Validated IKKa NF-kB Signaling Validated IKKb NF-kB Signaling Validated NF-kB1 NF-kB Signaling Cloned NF-kB2 NF-kB Signaling Cloned Bcl-3 NF-kB Signaling Cloned Calcineurin TCR/BCR Signaling Cloned NFATc1 TCR Signaling Cloned NFATc2 TCR Signaling Cloned NFATc3 TCR Signaling Cloned NFATc4 TCR Signaling Cloned Csp1 TCR/BCR Signaling Cloned Csp2 TCR/BCR Signaling Cloned Csp3 TCR/BCR Signaling Cloned Carma TCR/BCR Signaling Cloned Calm TCR/BCR Signaling Cloned Cabin TCR/BCR Signaling Cloned Genetics Core 2009-10-09T18:01:34+00:00 http://www.refaelilab.com/index.php/site/mouse_and_cell_lines/ http://www.refaelilab.com/index.php/site/mouse_and_cell_lines/#When:17:53:27Z Mouse Lines All of our mice are on a C57BL/6 background or are in the process of becoming so. We have several strains which overexpress MYC, either consitutively, in a B-cell or T-cell specific manner, or inducibly. We also have several mice for examining antigen receptors and their ligands. E-mu MYC - Transgenics, express MYC off of the heavy-chain Ig promoter, C57BL/6 background. Floxed MYC - Endogenous MYC allele flanked by loxP sites. Mixed Background. TRE-MYC - TET-responsive MYC,currently backcrossing to C57BL/6 HEL-VDJ-Knock-in - HEL-specific VDJ knocked-into the endogenous IgH locus. C57BL/6 background. BCR-HEL - Transgenics, C57BL/6 background. express a BCR specific for HEL MD4 - HEL specific BCR transgene, C57BL/6 ML5 - Express soluble HEL, C57BL/6 background. KLK4 - membrane-bound HEL, C57BL/6 background. OTII TCR - Transgenics, TCR specific for Kb and an Ova peptide. C57BL/6 background. MMTV-rtTA - B-cell specific TET-off repressor, currently backcrossing to C57BL/6 E-mu-rtTA - T-cell specific TET-off repressor, currently backcrossing to C57BL/6 YETI - YFP-enhanced transcript of IFN-gamma. C57BL/6 background. P110-Delta Knock-in - Knock-in Point mutation for PI3K-P110-delta isoform (inactivated). C57BL/6 background. Cell Lines We have many cell lines derived from the T and B cell lymphomas and leukemias isolated from the spleens, lymph nodes and thymi of many of our different transgenic mice. These include: E-mu-Myc/MD4 Eu-Myc/MD4/MLS MMTV-tTA/TRE-Myc/MD4 MMTV-tTA/TRE-Myc/MD4/MLS E-mu-tTA/TRE-Myc Myc-ER/hBL1-2 HSC’s Mouse Cell 2009-10-09T17:53:27+00:00 http://www.refaelilab.com/index.php/site/refaeli_lab_personnel/ http://www.refaelilab.com/index.php/site/refaeli_lab_personnel/#When:15:17:45Z Yosef Refaeli, Ph.D. Dr. Refaeli has over 18 years of experience in the areas of virology, molecular biology, protein chemistry, immunology, cancer research and genetic manipulation of hematopoietic stem cells. This background has given him extensive expertise in the areas of genetic manipulation of cells and regulation of gene expression and transformation in hematopoietic lineages. Dr. Refaeli graduated from the university of Pennsylvania with an A.B Degree in Biology (cum laude). While he was an undergraduate, Dr. Refaeli participated in research work in the laboratory of Dr. David Weiner, in the Department of Pathology at the Medical School there. The work was focused on the regulation of HIV gene expression with specific emphasis on the accessory gene called VPR. Dr. Refaeli’s work led to the biochemical demonstration that VPR interacts with the glucocorticoid receptor complex and affects gene expression from the viral LTR by hijacking the cellular machinery. That work was the initial data that lead to a Phase II clinical trial with inhibitors of Glucocorticoid receptor function to abrogate HIV infection. Dr. Refaeli went on to obtain his Ph.D at the Harvard Medical School under the supervision of Dr. Abul Abbas, in the Committee for Immunology. This work pertained to the regulation of cell survival and apoptosis of T-cells following antigenic stimulation in vivo. These studies lead to the development of a novel method to retrovirally transduce primary lymphoid cells and bone marrow derived hematopoietic stem cells in work that was done in collaboration with members of Dr. David Baltimore’ laboratory. Dr. Refaeli then went on to pursue postdoctoral studies under the supervision of Dr. J. Michael Bishop, at the University of California in San Francisco. The studies pursued there were focused on the contribution of signals derived from the antigen receptor and their cooperation with MYC in normal lymphoid homeostasis and malignant transformation. Dr. Refaeli was hired as an assistant professor at the National Jewish Medical and Research Center in October of 2003, where he established his research group and several novel research directions. In 2006, Dr. Refaeli co-founded Taiga Biotechnologies along with Dr. Turner. In April of 2009, Dr. Refaeli joined the Charles C. Gates Program in Regenerative Medicine and Stem Cell Biology at the University of Colorado Denver School of Medicine. Dr. Refaeli’s laboratory is currently housed at the AMC campus in Aurora. He currently holds secondary appointments in several departments at the University of Colorado, including the Integrated Department of Immunology, The Program of Molecular Biology as well as in the MD/PhD Training Program. Dr. Refaeli’s laboratory focuses on three broad areas: first, extending our understanding the contribution of antigen receptor signaling to lymphoid neoplasia, second, understanding the role of proto-oncogenes in the regulation of lymphoid tolerance and disease and third, exploring the role of MYC in the regulation of stem cell proliferation, survival, self-renewal and differentiation. The lab has been funded by grants awarded from the Leukemia and Lymphoma Society, the Cancer League of Colorado as well as the National Institutes of Health, National Cancer Institute, National Institute of Heart, Lung and Blood, as well as the National Institute of Allergy and Infectious Diseases. Brian C. Turner, Ph.D. Research Instructor, UCD Department of Dermatology Brian earned his B.A. in history from Cornell University in 1994. He then spent some time working in the Denver Department of Public Health where he developed an interest in that field. Brian then earned a master’s degree from the Bloomberg School of Public Health at the Johns Hopkins University in Baltimore. After working in Dr. David Schwartz’s laboratory at JHU, Brian developed an interest in basic biomedical research and in microbiology in particular. Brian then enrolled in the Biological Sciences Graduate Program at the University of Colorado Health Sciences. He then joined Dr. Kathryn Holmes’ laboratory where he earned his Ph.D. in studies related to the molecular determinants of murine coronavirus receptor specificity, CEACAM1. Used site-directed mutagenesis to identify residues involved in resistance and susceptibility to strains of MHV. Brian joined the Refaeli laboratory in 2004. Brian was promoted to the rank of Research Instructor in 2007. Gregory A. Bird, Ph. D. Postdoctoral Research Fellow Gregory received his undergraduate degree in molecular cellular and developmental biology from the University of Colorado at Boulder in 1995. In 1996 he began working for Cytokine Sciences as a RPA. In 1997 he went to work for Sangamo BioSciences. His job at Sangamo was to clone, express, and test recombinant Zinc-finger DNA binding proteins fused to a transcriptional activation or repression domain. Such factors are now in preclinical and clinical trials for diseases such as Diabetic Neuropathy, HIV/AIDS, and Cancer. In 1999 he began working on his Ph.D. in the laboratory of Dr. David Bentley at the University of Colorado Health Sciences Center. His research in Dr. Bentley’s lab focused on trying to understand the role the C-terminal domain of RPB1, the largest subunit of RNA polymerase II, plays in pre-mRNA processing and transcription termination. He earned his Ph.D. in 2005. In the spring of 2005 he took a postdoctoral fellowship in the laboratory of Dr. Bruce Banfield working on Alphaherpesvirus. During the course of his post-doc in the Banfield lab, they established a collaboration with the laboratory of Dr. Yosef Refaeli investigating the use of Alphaherpesesvirus as viral vectors for the treatment of Lymphoma. In 2008, he transitioned full-time into the Refaeli lab. Elizabeth Brazeau, Ph.D. Postdoctoral Research Fellow Elizabeth completed her undergraduate studies at the University of Washington in Seattle with a bachelor’s degree in anthropology in 1998.  After graduating, she changed her focus to biological sciences.  She worked as a technician in the genetics department and participated in studies mapping protein interactions using the yeast two-hybrid system in the Fields laboratory.  She became interested in infectious disease and began working in the microbiology department where she worked with Kaposi’s sarcoma herpes virus in the Lagunoff laboratory before applying to graduate school in 2003. She recently completed graduate school at the University of Colorado, Anschutz Medical Campus with a Ph.D. in microbiology from the Gilden laboratory where she studied varicella viruses, the causative agent of chickenpox and shingles.  Her graduate work analyzed the role of varicella zoster ORF 63 in the process of virus-induced apoptosis in culture.  She also worked with a simian varicella virus cosmid system in an attempt to generate a virus deleted for both copies of latency associated protein ORF 63, thought to be important for establishment and maintenance of latent infection.  Her studies determined that varicella predominantly uses the intrinsic pathway of apoptosis during viral infection.  She is excited to be starting a post doctoral fellowship in the Refaeli laboratory (and so happy to be done with graduate school).  Avital Polsky, B.S. Professional Research Assistant Avital grew up in Las Cruces, New Mexico, where she developed an appreciation for green chile cheese fries and spent the majority of her free time doing ballet, tap, and jazz from age three to twenty. In the last six years of dancing, she was a member of the Las Cruces Chamber Ballet and a teacher at the dance studio.  She continued dancing and teaching into her first three years of college at New Mexico State University (NMSU), and then moved to New York in the Fall of 2005 to study for one year at SUNY Stony Brook.  This was followed by a yearlong hiatus from academics working as a dental assistant in Brooklyn and volunteer at Beth Israel Hospital in Manhattan (these two years were wonderful, except for the lack of green chile).  She returned to NMSU in May of 2007 where she worked under Dr. James Kroger through August of 2008, when she received her BS in Biology and Minor in Philosophy.  Avital has been a PRA in the Refaeli lab since November of 2008 and intends to go to graduate school to continue her studies in the field of biology (she’ll pack plenty of green chile wherever she goes). Dianne Ashby Professional Research Assistant,Veterinary Technician Dianne obtained here veterinary training at the Bel-Rea institute of animal technology where she obtained a Vetereinary Technical degree in 2005. She then gained experience as a veterinary technician in the OLAR at the University of Colorado Health Sciences Center and subsequently at the Biological Resources Center at National Jewish Medical and Research Center. Dianne was designated a group leader at the NJMRC during her last year there. Dianne became a registered Assisatnt Lab Animal Technician in October of 2007 and a registered Lab Animal Technician in January of 2009. She joined our group in April 2009 during our transition to UCD. Ryan Bjordhal, B.S. Graduate Student, Immunology Graduate Program Personnel 2009-10-08T15:17:45+00:00 http://www.refaelilab.com/index.php/site/refaeli_lab_joins_university_of_colorado_denver/ http://www.refaelilab.com/index.php/site/refaeli_lab_joins_university_of_colorado_denver/#When:14:49:32Z On April 1, 2009, the Refaeli Lab joined the Charles C. Gates Program in Regenerative Medicine and Stem Cell Biology and the Department of Dermatology at the University of Colorado Denver (UCD). With this transition came our relocation to the main UCD medical campus in Aurora, CO, in the former Fitzsimmons military base. We are now housed in a brand new medical campus adjacent to several laboratories that work of stem cells of the skin, lung, retina and pancreas. In addition, our new research directions have already produced novel collaborations and synergies on ongoing efforts to understand somatic cell reprogramming re-differentiation and application in clinical settings. One additional aspect of our move that is very exciting to us is the close proximity to the BioSciences Park. This is a facility that is administered by the Fitzsimmons redevelopment authority that aims to facilitate the transfer of technologies developed in academic laboratories into commercial ventures. This approach to translating our findings on the bench into clinically relevant applications is one of the driving forces of our laboratory. The presence of the BioScience Park across the street will help ensure this transition in an efficient and productive manner. In addition, we stand to learn about new areas by continuous discussions with the people involved in the clinical development of technologies generated in academia. This is an interaction that is very exciting for us and will be beneficial for both parties. In fact, a number of technologies developed in our laboratory have already been licensed to companies for further development. Home News 2009-10-08T14:49:32+00:00 http://www.refaelilab.com/index.php/site/ongoing_research_projects/ http://www.refaelilab.com/index.php/site/ongoing_research_projects/#When:20:37:56Z Question: What is the molecular basis of the interaction between antigen receptor signaling and dysregulated oncogenes? Answer: Understanding the mechanistic details that underlie the interaction between antigen receptor signals and dysregulated oncogenes may allow us to identify novel targets for the development of therapeutics for Non-Hodgkin’s lymphomas. We will address this issue by answering five interrelated questions. Question: Can other oncogenes also cooperate with chronic BCR signals? Answer: The experiments that the overexpression of MYC in the context of chronic BCR signals yielded a Burkitt’s-like (BL) tumor. Those neoplasms usually carry a translocation that juxtaposes the MYC locus to the Ig heavy or light chain genes (t8:14, or t8:22). We will test whether other oncogenes that have been identified in chromosomal translocations involving the Ig loci can also cooperate with continuous BCR signaling. These genes will be selected based on their ability to affect either cell proliferation (e.g., Bcl-1/CyclinD1), cell survival (e.g. Bcl-3/IkB-like), or both (e.g. Rel-NRG, or abl). The hope here is to broaden the conclusion and establish chronic BCR signaling as a common cooperating event in lymphoid neoplasia. Question: What is the nature of the signals derived from the BCR that cooperate with elevated levels of MYC? Answer: Our Burkitt’s-like mouse model also showed that commonly used immunosuppressive drugs were able to inhibit tumor formation, and lead to tumor regression. We will first confirm that the known protein targets of these drugs (e.g., Calcineurin for CsA and FK506, and mTOR for Rapamycin) in T cells are being affected in the transformed B cells in the BL-like tumors. Once these known targets are confirmed, we plan to construct retroviral vectors that encode dominant negative forms of these proteins, in bicistronic vectors that also express GFP. The idea is to retrovirally transduce tumor cells in vitro and transplant these into syngeneic mice. The ability of these cells to compete with their nontransduced counterparts will be used as a measure of the effect of these molecules to block the BCR signals that cooperate with MYC. In addition, we plan to analyze the effect of altering the activities of molecules that are acting upstream, or downstream of these known targets. This initial set of experiments will indicate whether the molecules in question are necessary for the BCR dependent signal to cooperate with MYC. One additional set of experiments we plan to pursue is to generate retroviruses that express constitutively active forms of the signaling molecules that appear to be necessary for the BCR dependent signal that cooperates with MYC. These viruses will be used to transduce B cells from Eμ—MYC mice that express a transgenic BCR on their surface, and transplant these into syngeneic mice. These experiments aim to define whether the molecules in question are sufficient to mediate the BCR signal that cooperates with MYC in lymphomagenesis. Along these lines, and in collaboration with Dr. Kenneth Field, we are currently testing the ability of dominant negative and constitutive active forms of small GTP-binding proteins (Ras, Rho, Rap) in BCR dependent oncogenesis. We also plan to examine whether these molecules can affect the proliferation or survival of cell lines derived from human Burkitt’s lymphoma tumors. One additional aspect of this set of experiments is the role of costimulatory and negative regulators of antigen receptor signaling. We plan to initially use inhibitory antibodies to cell surface molecules that participate in antigen receptor signaling (e.g. CD28 for T cells, and CD23 for B cells), or to negative regulators of the antigen receptor signals (e.g. CTLA4 for T cells, and FC?RII for B cells). The initial screen with antibodies will be followed up by genetic experiments involving the breeding of knockout alleles to our triple transgenic combination. The hope here is to identify specific signaling mediators that mediate the functional cooperation of BCR dependent signals with MYC. There is a chance that many molecules may qualify for this category, which broadens the number of potential targets we can further pursue for the development of small molecule inhibitors. Question: Studies on the biochemical nature of tonic BCR signals that cooperate with elevated levels of MYC. Answer: The nature of the signals that are involved in tonic signaling by the BCR is a largely understudied area in immunology, but one which may prove to be of great potential for the identification of drug targets for lymphoid tumors. We plan to use some of the same approaches described earlier for the analysis of the cognate antigen triggered signals involved in the tumorigenic cooperation with MYC, with two important differences. We plan to develop lentiviral constructs to transduce naive, resting B cells. This method of genetic manipulation will allow us to study the signaling requirements for naive B cell survival and maintenance of quiescence in vivo. These studies may also unveil potential molecules that can prevent the onset of cognate antigen triggered B cell activation. Question: Does the interaction between a dysregulated oncogene and chronic antigen receptor signaling also occur in T cells? Answer: One prediction from our results in B cells is that a similar interaction may be important for the development of T cell tumors. We are currently introducing T cell receptor (TCR) transgenes into a tetracycline-regulated MYC background. In analogy to the experiments we have pursued in the B cell compartment, the TCR transgene will fix the antigenic specificity of the majority of the T cells in these mice, and render them susceptible to stimulation by a known antigen. The antigen can be introduced by immunization, or transgenesis. The biological differences between T and B cells may turn out to be important for determining the parameters for an interaction with dysregulated oncogenes. These differences can potentially unveil some important differences in the nature of the molecules needed to target in the two compartments. Question: Does the timing of oncogene dysregulation alter the nature of the tumor? Answer: There are many B cell malignancies that appear to be antigen dependent (e.g. DLBCL, follicular B cell lymphoma, mantle cell lymphoma, etc). In order to test whether the key difference among these tumors is the time at which the oncogenes become dysregulated, We plan to breed the BCR-HEL and s-HEL transgenes onto a tetracytline-regulated background, that over expresses MYC in a B cell specific manner. These mice will be useful for determining whether the timing at which MYC is overexpressed alters the nature of the tumors obtained. They are also useful for determining whether the overexpression of MYC is required for maintaining these tumors, or alternatively, study the details underlying tumor regression. Question: What is the nature of the signals that mediate the proliferation of lymphocytes by MYC, downstream of cytokine receptors? Answer: Understanding the biochemical pathway by which cytokine receptors result in the regulation of MYC function may allow us to establish functional associations between MYC and other cellular protoconcogenes. Such functional interactions may provide some insights into treatment modalities for tumors with associated mutations. Question: What is the nature of the genes that are transcriptionally regulated by MYC in lymphocytes? Answer: We will compare the transcriptional profiles of primary lymphocytes that will be obtained from either mice that overexpress MYC in a tetracytcline-regulated fashion, or can delete their MYC loci by Cre/LoxP directed methods. These two sets of data may allow us to gain some perspective of the nature of the genes involved in MYC induced lymphocyte proliferation and survival. Candidate genes will be tested for their ability to replace MYC, in their constitutively active form, by transducing these genes into T or B cells obtained from mice in which the MYC loci is flanked by loxP sites, and can inducibly express Cre (Mx-Cre transgenic). If the genes are able to replace the proliferative or survival function of MYC, the transduced lymphocytes should behave different than their non-transduced counterparts. In addition, the transduction of T or B cells from tetracycline-regulated MYC overexpressing mice with dominant negative forms of these candidate genes may allow us to confirm their involvement in the MYC dependent proliferative and survival pathways. This system may also allow us to test whether these proto-oncogenes can lead to the development of tumors in a MYC independent fashion in vivo. Question: Does MYC regulate proliferation and survival in IL-2 family cytokines, or in a wide range on growth factors? Answer: We are interested in determining whether MYC is a key determinant of proliferation and survival in different cytokines. This is important to determine in the context of rendering transformed cells cytokine independent in the process of tumorigenesis. In collaboration with Dr. Warren Pear, Dr. Refaeli has shown that MYC is a critical regulator of Notch-1 dependent proliferation and survival in T cells, and is a negative regulator of Notch-1 dependent apoptosis in B cells. Further work in this area will attempt to determine if the molecular signals linking MYC to various cytokine receptors is common across different receptor families. Question: What is the role of host-inflammatory pathways in the establishment and maintenance of lymphoid malignancies? Answer: Dr. Refaeli’s work has shown that IFN-g is important for the development of tumor associated clinical signs in our BL-like tumor model. In addition, Dr. Refaeli has shown that IRF-1, a transcription factor that regulates IFN-type I (a/b) expression, is critical for the engraftment of BL-like tumors in vivo. We plan to further explore the requirements of inflammation for the initiation, establishment and maintenance of lymphoid malignancies in vivo. We have obtained mice in which the genes encoding the type I IFN receptor (INF a/b Receptor b), or type II IFN receptor (IFN g Receptor b) have been disrupted. We plan to breed these mutations onto our triple transgenic combination to further dissect the role or inflammatory mediators in lymphomagenesis. In addition, we plan to compare these experiments to tumor transplantation experiments. Along these lines, we have also used Cox-2 inhibitory drugs to explore the role of the downstream effectors of inflammation in tumor engraftment. The preliminary experiments Dr. Refaeli has conducted show an important role for Cox-2 in the engraftment of BL like tumors in vivo. We plan to further pursue this line of experiments with Cox-1 and Cox-2 deficient mice. In addition, the downstream targets of Cox-2 include VEGF. A potential relationship between inflammation and angiogenic events may turn out to be an important source of new targets for the prophylactic treatment of lymphoid tumors. Projects 2009-09-11T20:37:56+00:00 http://www.refaelilab.com/index.php/site/lentiviral_infection/ http://www.refaelilab.com/index.php/site/lentiviral_infection/#When:19:41:08Z Lentiviral Infection of T, B and BM Cells Media C10: Lymphocytes: RPMI 1640 + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME D10: Lymphocytes: DMEM + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME Bone Marrow: DMEM + 15% FCS, L-glu, pen/strep, IL-3, IL-6, SCF Day 1 Plate 0.7x106 293FT cells in a 60mm dish in 4ml of D10 media Day 2 In the early evening, begin transfection For each infection, place 150 Buffer EC into a siliconized 1.5 mL eppendorf tube. Add 0.5ug of PI, PII and PIII packaging vectors. Do not premix packaging vectors. Add 1.0ug of viral plasmid (pLL3.7, etc) Gently mix Add 16ul of Enhancer Gently mix, wait 5’ Add 32ul of Effectene Gently mix, wait 5’ Add to plated 293FT’s from day 1. Gently drip into media. Incubate at 37 C. Protocols 2009-07-23T19:41:08+00:00 http://www.refaelilab.com/index.php/site/lentiviral_cloning/ http://www.refaelilab.com/index.php/site/lentiviral_cloning/#When:19:36:38Z Lentiviral shRNA Cloning Protocol (pLL3.7, 3.72, 3.77, pSICO, pSICOR) Oligo Preparation 1ul Sense Oligo 1ul anti-sense oligo 48ul Annealing Buffer (100mM K-acetate, 30mM HEPES-KOH (7.4), 2mM Mg-acetate) Either use PCR machine to heat to 95 C for 10’, and then slowly cool to RT, or heat in heatblock for 10’ and then remove from heating unit and allow to cool to RT. Vector Preparation Digest 5-10ug of vector DNA with Xho1 and Hpa1 for 3 hr at 37 C Add 1ul per rxn of CIP, incubate at 37 C for 30’ Quickly run rxn after CIP incubation on 1% agarose gel at 180V for ~45 min Purify cut vector band via Qiaex Kits Protocols 2009-07-23T19:36:38+00:00 http://www.refaelilab.com/index.php/site/retroviral_infection/ http://www.refaelilab.com/index.php/site/retroviral_infection/#When:19:32:56Z Media C10: Lymphocytes: RPMI 1640 + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME D10: Lymphocytes: DMEM + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME Bone Marrow Media: DMEM + 15% FCS, L-glu, pen/strep, IL-3, IL-6, SCF Preparing T cells for Infection Plate 106 purified CD4+ T cells Activate with 1ug/ml of anti-CD3 and anti-CD28 Infect after 24hrs Preparing B cells for Infection Plate 106 purified CD4+ T cells Activate with 1ug/ml of anti-IgM and anti-CD40 Infect after 24hrs Protocols 2009-07-23T19:32:56+00:00 http://www.refaelilab.com/index.php/site/retroviral_cloning/ http://www.refaelilab.com/index.php/site/retroviral_cloning/#When:19:04:25Z Cloning of Retroviral Vectors (pMIG) Digesting Plasmid Digest 3ug of DNA to a final volume of 60ul, for 4 hours Before the last hour of digestion, 1ul of CIP To generate blunt ends: at RT add 2ul of 10mM dNTPs, and 2ul of Klenow Incubate for 20 minutes at RT Immediately run on a 1% low melting agarose gel Ligation Add 11ul of insert and 4ul of vector 4ul of buffer (either NEAB, or Gibco; with PEG) 1ul of NEB high conc. Ligase (2,000,000 u/ml) Incubate overnight at 16ºC Add 80ul of water, 40ul of NaOAc (3M), and 300ul of EtOH Spin for 10 minutes at 14k; wash 1x in 70% EtOH Resuspend in 5ul of water, use entire volume to transform Nova-blue bugs. Protocols 2009-07-23T19:04:25+00:00