370 research outputs found
Interview with the Daughters of Marjorie Faucett Patterson
https://digitalcommons.winthrop.edu/scmotheroftheyear/1010/thumbnail.jp
Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.
One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA
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Molecular characterization of the MX genes of rainbow trout (Oncorhynchus mykiss)
The Mx protein family of rainbow trout (Oncorhynchus mykiss) was characterized at the molecular level. With primers derived from a partial sequence of Perch (Perca fluviatis) Mx genomic DNA, a partial rainbow trout Mx genomic DNA sequence was obtained by polymerase chain reaction (PCR) amplification. All salmonid fish investigated contained Mx homologous DNA sequences as determined by Southern blot. Northern blot analysis of Mx expression demonstrated that Mx mRNA was induced by poly inosinic cytidylic (IC) dsRNA both in vitro and in vivo and by infectious hematopoietic necrosis virus (IHNV) in vivo. RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) was used to clone three Mx cDNA clones (designated RBTMx) from the rainbow trout gonad cell line, RTG-2, after induction by poly IC dsRNA. The deduced RBTMx 1, 2, and 3 proteins are 621, 636, and 623 amino acids in length with molecular weights of 70.6, 72, and 70.8 kD respectively. The trout Mx proteins share approximately 50% identity at the amino acid level with mammalian and avian Mx proteins and contain the tripartite GTP binding domain and leucine zipper motifs common to all Mx proteins.
Polyclonal antisera generated to an E. coli expressed trout Mx protein fragment recognized all three trout Mx proteins. Poly IC dsRNA induced the expression of trout Mx protein in vitro. Mx protein was detected in the liver and kidney of rainbow trout challenged with IHNV. Immunohistochemical staining demonstrated that Mx protein was expressed in the kidney tubules of rainbow trout following exposure to IHNV.
Trout Mx cDNA clones were transiently expressed under the control of a eukaryotic viral promoter in a salmon embryo cell line by transfection. RBTMx1 was expressed in the cytoplasm in regions surrounding the nucleus. Staining for RBTMx2 produced a punctate pattern within the nucleus, characteristic of mammalian and rodent Mx proteins, and in the cytoplasm of some cells. RBTMx3 was expressed in a diffuse pattern, uniformly distributed throughout the cytoplasm, but not within the nucleus. Transient expression of the trout Mx proteins did not result in inhibition of II-INV replication as evidenced by nucleocapsid protein accumulation
Targeted imaging of colorectal dysplasia in living mice with fluorescence microendoscopy
We validate specific binding activity of a fluorescence-labeled peptide to colorectal dysplasia in living mice using a miniature, flexible, fiber microendoscope that passes through the instrument channel of an endoscope. The microendoscope delivers excitation light at 473 nm through a fiber-optic bundle with outer diameter of 680 Āµm to collect en face images at 10 Hz with 4 Āµm lateral resolution. We applied the FITC-labeled peptide QPIHPNNM topically to colonic mucosa in genetically engineered mice that spontaneously develop adenomas. More than two-fold greater fluorescence intensity was measured from adenomas compared to adjacent normal-appearing mucosa. Images of adenomas showed irregular morphology characteristic of dysplasia
A novel approach to identify driver genes involved in androgen-independent prostate cancer
Abstract
Background
Insertional mutagenesis screens have been used with great success to identify oncogenes and tumor suppressor genes. Typically, these screens use gammaretroviruses (Ī³RV) or transposons as insertional mutagens. However, insertional mutations from replication-competent Ī³RVs or transposons that occur later during oncogenesis can produce passenger mutations that do not drive cancer progression. Here, we utilized a replication-incompetent lentiviral vector (LV) to perform an insertional mutagenesis screen to identify genes in the progression to androgen-independent prostate cancer (AIPC).
Methods
Prostate cancer cells were mutagenized with a LV to enrich for clones with a selective advantage in an androgen-deficient environment provided by a dysregulated gene(s) near the vector integration site. We performed our screen using an in vitro AIPC model and also an in vivo xenotransplant model for AIPC. Our approach identified proviral integration sites utilizing a shuttle vector that allows for rapid rescue of plasmids in E. coli that contain LV long terminal repeat (LTR)-chromosome junctions. This shuttle vector approach does not require PCR amplification and has several advantages over PCR-based techniques.
Results
Proviral integrations were enriched near prostate cancer susceptibility loci in cells grown in androgen-deficient medium (pā<ā0.001), and five candidate genes that influence AIPC were identified; ATPAF1, GCOM1, MEX3D, PTRF, and TRPM4. Additionally, we showed that RNAi knockdown of ATPAF1 significantly reduces growth (pā<ā0.05) in androgen-deficient conditions.
Conclusions
Our approach has proven effective for use in PCa, identifying a known prostate cancer gene, PTRF, and also several genes not previously associated with prostate cancer. The replication-incompetent shuttle vector approach has broad potential applications for cancer gene discovery, and for interrogating diverse biological and disease processes.http://deepblue.lib.umich.edu/bitstream/2027.42/109477/1/12943_2014_Article_1323.pd
Novel reporter systems for facile evaluation of I-SceI-mediated genome editing
Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects
Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template
Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI ācleavaseā, but no evidence of toxicity with the I-AniI ānickase.ā Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs
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