Comparison of lentiviral vector titration methods

Geraerts, Martine; Willems, S.M.; Baekelandt, Veerle; Debyser, Zeger; Gijsbers, Rik

doi:10.1186/1472-6750-6-34

Cited by 137 publications

(104 citation statements)

References 27 publications

(47 reference statements)

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“…Follow the procedure described in steps 3–6 in Subheading 3.2.1 and, after 48-h selection, determine the amount of lentivirus required to obtain ~60 % of puromycin (or other mammalian plasmid selection)-resistant cells. If quantitative determination of lentivirus titer is desired, follow one of the protocols described by Geraerts et al [15]. …”

Section: Methodsmentioning

confidence: 99%

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation

Tapia

Gerace

2016

Methods in Molecular Biology

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We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.

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Section: Methodsmentioning

confidence: 99%

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation

Tapia

Gerace

2016

Methods in Molecular Biology

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“…Collected media were pooled and centrifuged at 40,000 x g and the viral pellet was suspended in a small volume of PBS. DDR2 shRNA or control shRNA viral particles titration analysis in the presence of 4 μg/ml puromycin indicated an optimal dose of 19 × 10 8 transduction units/ml (TU/ml) for 1 × 10 6 primary osteoblasts [25]. In knockdown experiments, primary osteoblasts were transduced with DDR2 shRNA or control shRNA at 80–90% visual confluence for 24 hours.…”

Section: Methodsmentioning

confidence: 99%

Collagen advanced glycation inhibits its Discoidin Domain Receptor 2 (DDR2)-mediated induction of lysyl oxidase in osteoblasts

Khosravi

Sodek

Faibish

et al. 2014

Bone

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Diabetes increases the risk of bone fracture. Organic and inorganic bone extracellular matrix components determine bone strength. Previous studies indicate that in diabetes, glycation of collagen causes abnormal arrangements of collagen molecules and fragile bones. Diabetic bone fragility is additionally attributed to reduced levels of lysyl oxidase enzyme-dependent collagen cross-links. The mechanism underlying the presence of lower enzymatic collagen cross-links in diabetic bone has not been directly investigated. Here we determine in primary osteoblast cultures the regulation of lysyl oxidase protein by type I collagen and collagen modified by carboxymethylation (CML-collagen), a form of advanced glycation endproducts. Data indicate that non-glycated collagen up-regulates lysyl oxidase levels both in primary non-differentiated and in differentiating mouse and rat osteoblast cultures, while CML-collagen fails to regulate lysyl oxidase in these cells. Collagen binding to Discoidin Domain Receptor-2 (DDR2) mediates lysyl oxidase increases, determined in DDR2 shRNA knockdown studies. DDR2 binding and activation were disrupted by collagen glycation, pointing to a mechanism for the diminished levels of lysyl oxidase and consequent low lysyl oxidase-derived cross-links in diabetic bone. Our studies indicate that collagen-integrin interactions may not play a major role in up-regulating lysyl oxidase. Furthermore, non-collagenous ligands for the receptor for advanced glycation end products (RAGE) failed to alter lysyl oxidase levels. Taken together with published studies a new understanding emerges in which diabetes- and age-dependent inhibition of normal collagen-stimulated DDR2- and integrin-signaling, and independent advanced glycation-stimulated RAGE-signaling, each contributes to different aspects of diabetic osteopenia.

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“…32,33 The next day, the transduced cells were infused into rhesus macaques without conditioning. The transduction rates were evaluated by %GFP in flow cytometry and VCN in real-time PCR using WPRE-specific probe/primers, 34 which can detect the GFP/WPRE-transduced lymphocytes (from lymphocyte infusion), but not blood cells from GFP-transduced HSCs (from stem cell transplantation).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model

et al. 2013

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Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34+ cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34+ cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.

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Comparison of lentiviral vector titration methods

Cited by 137 publications

References 27 publications

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation

Collagen advanced glycation inhibits its Discoidin Domain Receptor 2 (DDR2)-mediated induction of lysyl oxidase in osteoblasts

Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model

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