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Results: 1 to 10 of 46

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CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line.
Veach RA, Wilson MH
(2018) PLoS One 13: e0204487
MeSH Terms: Acute Kidney Injury, CRISPR-Cas Systems, Cell Line, Cisplatin, Gene Knock-In Techniques, Gene Targeting, Genes, Reporter, Genetic Engineering, Glucose, Green Fluorescent Proteins, Hepatitis A Virus Cellular Receptor 1, Homologous Recombination, Humans, Kidney Tubules, Proximal, Luciferases, Up-Regulation
Show Abstract · Added December 13, 2018
We used the CRISPR/Cas9 system to knock-in reporter transgenes at the kidney injury molecule-1 (KIM-1) locus and isolated human proximal tubule cell (HK-2) clones. PCR verified targeted knock-in of the luciferase and eGFP reporter at the KIM-1 locus. HK-2-KIM-1 reporter cells responded to various stimuli including hypoxia, cisplatin, and high glucose, indicative of upregulation of KIM-1 expression. We attempted using CRISPR/Cas9 to also engineer the KIM-1 reporter in telomerase-immortalized human RPTEC cells. However, these cells demonstrated an inability to undergo homologous recombination at the target locus. KIM-1-reporter human proximal tubular cells could be valuable tools in drug discovery for molecules inhibiting kidney injury. Additionally, our gene targeting strategy could be used in other cell lines to evaluate the biology of KIM-1 in vitro or in vivo.
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16 MeSH Terms
Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.
Messer WB, Yount BL, Royal SR, de Alwis R, Widman DG, Smith SA, Crowe JE, Pfaff JM, Kahle KM, Doranz BJ, Ibarra KD, Harris E, de Silva AM, Baric RS
(2016) J Virol 90: 5090-5097
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Cross Reactions, Dengue, Dengue Virus, Disease Models, Animal, Epitopes, Genetic Engineering, Humans, Mice, Neutralization Tests, Serogroup
Show Abstract · Added May 4, 2016
UNLABELLED - The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms.
IMPORTANCE - Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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14 MeSH Terms
piggyBac-ing models and new therapeutic strategies.
Woodard LE, Wilson MH
(2015) Trends Biotechnol 33: 525-33
MeSH Terms: Animals, Baculoviridae, Genetic Engineering, Genetic Vectors, Genome, Humans, Mice
Show Abstract · Added July 28, 2015
DNA transposons offer an efficient nonviral method of permanently modifying the genomes of mammalian cells. The piggyBac transposon system has proven effective in genomic engineering of mammalian cells for preclinical applications, including gene discovery, simultaneous multiplexed genome modification, animal transgenesis, gene transfer in vivo achieving long-term gene expression in animals, and the genetic modification of clinically relevant cell types, such as induced pluripotent stem cells and human T lymphocytes. piggyBac has many desirable features, including seamless excision of transposons from the genomic DNA and the potential to target integration events to desired DNA sequences. In this review, we explore these recent applications and also highlight the unique advantages of using piggyBac for developing new molecular therapeutic strategies.
Published by Elsevier Ltd.
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7 MeSH Terms
Revisiting the case for genetically engineered mouse models in human myelodysplastic syndrome research.
Zhou T, Kinney MC, Scott LM, Zinkel SS, Rebel VI
(2015) Blood 126: 1057-68
MeSH Terms: Animals, Disease Models, Animal, Genetic Engineering, Hematopoiesis, Hematopoietic Stem Cells, Humans, Mice, Myelodysplastic Syndromes
Show Abstract · Added April 25, 2016
Much-needed attention has been given of late to diseases specifically associated with an expanding elderly population. Myelodysplastic syndrome (MDS), a hematopoietic stem cell-based blood disease, is one of these. The lack of clear understanding of the molecular mechanisms underlying the pathogenesis of this disease has hampered the development of efficacious therapies, especially in the presence of comorbidities. Mouse models could potentially provide new insights into this disease, although primary human MDS cells grow poorly in xenografted mice. This makes genetically engineered murine models a more attractive proposition, although this approach is not without complications. In particular, it is unclear if or how myelodysplasia (abnormal blood cell morphology), a key MDS feature in humans, presents in murine cells. Here, we evaluate the histopathologic features of wild-type mice and 23 mouse models with verified myelodysplasia. We find that certain features indicative of myelodysplasia in humans, such as Howell-Jolly bodies and low neutrophilic granularity, are commonplace in healthy mice, whereas other features are similarly abnormal in humans and mice. Quantitative hematopoietic parameters, such as blood cell counts, are required to distinguish between MDS and related diseases. We provide data that mouse models of MDS can be genetically engineered and faithfully recapitulate human disease.
© 2015 by The American Society of Hematology.
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8 MeSH Terms
CRISPR-Cas9 knockin mice for genome editing and cancer modeling.
Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F
(2014) Cell 159: 440-55
MeSH Terms: Adenocarcinoma, Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Dendritic Cells, Disease Models, Animal, Gene Knock-In Techniques, Genes, Tumor Suppressor, Genetic Engineering, Genetic Vectors, Lentivirus, Lung Neoplasms, Mice, Mice, Transgenic, Oncogenes
Show Abstract · Added September 7, 2016
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
Copyright © 2014 Elsevier Inc. All rights reserved.
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14 MeSH Terms
Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia.
Saito S, Nakazawa Y, Sueki A, Matsuda K, Tanaka M, Yanagisawa R, Maeda Y, Sato Y, Okabe S, Inukai T, Sugita K, Wilson MH, Rooney CM, Koike K
(2014) Cytotherapy 16: 1257-69
MeSH Terms: Antigens, CD19, Cancer Vaccines, Cell Line, Tumor, Cell Proliferation, Culture Media, Serum-Free, Cytotoxicity, Immunologic, DNA Transposable Elements, Drug Resistance, Neoplasm, Genetic Engineering, Genetic Vectors, Humans, Immunotherapy, Adoptive, Interleukin-15, Interleukin-2, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Mutation, Protein Kinase Inhibitors, Receptors, Antigen, T-Cell, Recombinant Fusion Proteins, T-Lymphocytes, TNF-Related Apoptosis-Inducing Ligand, Up-Regulation
Show Abstract · Added October 28, 2014
BACKGROUND AIMS - To develop a treatment option for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR).
METHODS - A CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15-containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph(+)ALL cell lines including three TKI-resistant (T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines.
RESULTS - We obtained ∼1.3 × 10(8) CAR T cells (CD4(+), 25.4%; CD8(+), 71.3%), co-expressing CD45RA and CCR7 up to ∼80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CAR T cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor-related apoptosis-inducing ligand and interleukin-2.
CONCLUSIONS - We generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac-transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph(+)ALL regardless of T315I mutation. PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph(+)ALL.
Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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22 MeSH Terms
Genome engineering using the CRISPR-Cas9 system.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
(2013) Nat Protoc 8: 2281-2308
MeSH Terms: Base Sequence, Cell Culture Techniques, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, DNA End-Joining Repair, DNA Mutational Analysis, DNA Repair, Deoxyribonucleases, Genetic Engineering, Genome, Genotyping Techniques, HEK293 Cells, Humans, Molecular Sequence Data, Mutagenesis, Transfection
Show Abstract · Added February 10, 2015
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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16 MeSH Terms
Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.
Jao LE, Wente SR, Chen W
(2013) Proc Natl Acad Sci U S A 110: 13904-9
MeSH Terms: Animals, Breeding, Deoxyribonucleases, Gene Knockout Techniques, Genetic Engineering, Genome, Inverted Repeat Sequences, Mutagenesis, Site-Directed, Phenotype, Zebrafish
Show Abstract · Added March 7, 2014
A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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2 Members
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10 MeSH Terms
Multiplex genome engineering using CRISPR/Cas systems.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F
(2013) Science 339: 819-23
MeSH Terms: Animals, Base Sequence, CRISPR-Cas Systems, DNA, DNA Cleavage, Genetic Engineering, Genetic Loci, Genome, Humans, Inverted Repeat Sequences, Mice, Microarray Analysis, Molecular Sequence Data, Mutagenesis, RNA, Recombinational DNA Repair, Streptococcus pyogenes
Show Abstract · Added August 13, 2013
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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17 MeSH Terms
Comparative analysis of the recently discovered hAT transposon TcBuster in human cells.
Woodard LE, Li X, Malani N, Kaja A, Hice RH, Atkinson PW, Bushman FD, Craig NL, Wilson MH
(2012) PLoS One 7: e42666
MeSH Terms: Animals, Base Sequence, DNA Primers, DNA Transposable Elements, Gene Transfer Techniques, Genetic Engineering, HEK293 Cells, Humans, INDEL Mutation, Molecular Sequence Data, Mutagenesis, Insertional, Plasmids, Transposases, Tribolium
Show Abstract · Added August 22, 2013
BACKGROUND - Transposons are useful tools for creating transgenic organisms, insertional mutagenesis, and genome engineering. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes.
METHODOLOGY/PRINCIPAL FINDINGS - We tested TcBuster for its activity in human embryonic kidney 293 (HEK-293) cells. Excision footprints obtained from HEK-293 cells contained small insertions and deletions consistent with a hAT-type repair mechanism of hairpin formation and non-homologous end-joining. Genome-wide analysis of 23,417 piggyBac, 30,303 Sleeping Beauty, and 27,985 TcBuster integrations in HEK-293 cells revealed a uniquely different integration pattern when compared to other transposon systems with regards to genomic elements. TcBuster experimental conditions were optimized to assay TcBuster activity in HEK-293 cells by colony assay selection for a neomycin-containing transposon. Increasing transposon plasmid increased the number of colonies, whereas gene transfer activity dependent on codon-optimized transposase plasmid peaked at 100 ng with decreased colonies at the highest doses of transposase DNA. Expression of the related human proteins Buster1, Buster3, and SCAND3 in HEK-293 cells did not result in genomic integration of the TcBuster transposon. TcBuster, Tol2, and piggyBac were compared directly at different ratios of transposon to transposase and found to be approximately comparable while having their own ratio preferences.
CONCLUSIONS/SIGNIFICANCE - TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.
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14 MeSH Terms