Production and purification of lentiviral vectors

Tiscórnia, Gustavo; Singer, Oded; Verma, Inder M.

doi:10.1038/nprot.2006.37

Cited by 881 publications

(683 citation statements)

References 16 publications

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“…pFUmChW (mCherry) was made by replacing GFP by mCherry in the pFUGW vector and has been described before 7 . Lentiviral particles were produced according to a protocol adapted from 23,24 . Briefly, the lenviral construct of interest was co-transfected in HEK293T cells together with the envelope (VSV-G) and packaging (Δ8.9) vectors.…”

Section: Dna Constructs Lentiviruses and Antibodiesmentioning

confidence: 99%

STIM2 regulates AMPA receptor trafficking and plasticity at hippocampal synapses

Yap

Shetty

García-Alvarez

et al. 2017

Neurobiology of Learning and Memory

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STIM2 is an integral membrane protein of the endoplasmic reticulum (ER) that regulates the activity of plasma membrane (PM) channels at ER-PM contact sites. Recent studies show that STIM2 promotes spine maturation and surface expression of the AMPA receptor (AMPAR) subunit GluA1, hinting to a probable role in synaptic plasticity. Here, we used a Stim2 cKO mouse line to explore the function of STIM2 in early forms of Long-Term Potentiation (E-LTP) and Depression (E-LTD), two widely-studied models of synaptic plasticity implicated in information storage. We found that STIM2 is required for the stable expression of both E-LTP and E-LTD at CA3-CA1 hippocampal synapses. Altered plasticity in Stim2 cKO mice is associated with subtle alterations in the shape and density of dendritic spines in CA1 neurons.Further, surface delivery of GluA1 in response to LTP-inducing chemical manipulations was markedly reduced in excitatory neurons derived from Stim2 cKO mice. In addition, NMDAinduced GluA1 endocytosis, which underlies LTD, was impaired in Stim2 cKO neurons. We conclude that STIM2 facilitates synaptic delivery and removal of AMPARs and regulates activity-dependent changes in synaptic strength through a unique mode of communication between the ER and the synapse.

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Section: Dna Constructs Lentiviruses and Antibodiesmentioning

confidence: 99%

STIM2 regulates AMPA receptor trafficking and plasticity at hippocampal synapses

Yap

Shetty

García-Alvarez

et al. 2017

Neurobiology of Learning and Memory

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“…12,13 As the lentivirus carries green fluorescence protein (GFP), the viral titer was determined by counting GFP-expressing cells under fluorescence microscopy 96 h after infection as described in previous reports. 14 …”

Section: Construction Of Zfx Short Harpin Rna-expressing Lentivirusmentioning

confidence: 99%

Knockdown of zinc finger protein X-linked inhibits prostate cancer cell proliferation and induces apoptosis by activating caspase-3 and caspase-9

et al. 2012

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Zinc finger protein X-linked (ZFX) is a highly conservative mammalian gene with related functions in cell survival and proliferation. However, there are limited reports on regulation of ZFX as a therapeutic target in cancer treatment. In this study, the expression of ZFX in prostate cancer with matched normal adjacent tissues (n ¼ 45) and benign prostatic hyperplasia (BPH) tissues (n ¼ 16) were observed by immunohistochemical analysis. The effect of lentiviral siRNA (small interference RNA)-mediated dysfunction of ZFX on the proliferation of prostate cancer cells was studied. ZFX mRNA and protein expression levels in prostate cancer cells (PC-3 and DU145) were analyzed by western blotting and real-time polymerase chain reaction (RT-PCR). The effects of siRNA targeting ZFX on growth, cell cycle and apoptosis of PC-3 cells were evaluated by MTT assay and flow cytometry. We also investigated the effect of ZFX deletion on the activation of caspase-1, -3 and -9 by western blotting and colorimetric assay. Prostate cancer specimens appeared significantly higher (42.2% of cases being positive) than that observed in normal adjacent tissues (11.8% of cases being positive) and BPH tissues (12.5% of cases being positive). Repression of ZFX in the prostate cancer cells effectively suppressed the cellular proliferation and colony-formation ability, and led to G1 phase cell cycle arrest. Moreover, inhibition of ZFX-induced cell apoptosis by activating caspase-1, -3 and -9. In conclusion, ZFX represents the prominent role in the progression of prostate cancer and may be a promising therapy target for prostate cancer.

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“…The GFP-expressing pCSC-SP-PW-IRES/GFP empty vector was used as control. Recombinant lentiviruses were produced by co-transfecting HEK293T cells with the Smad expression or control transfer plasmid and three additional plasmids required for packaging (pMDL, pRev, and pVSVG) using polyethylenimine as the transfection reagent, as described (44). The supernatants containing the viral particles were collected 48 h after transfection, filtered through a 0.45-m filter, and concentrated by ultracentrifugation for 2.5 h at 50,000 ϫ g. Relative titers were assessed by monitoring the percentage of GFP-positive HEK293T cells infected with serial dilutions of the viral preparations.…”

Section: Methodsmentioning

confidence: 99%

A Smad-binding Element in Intron 1 Participates in Activin-dependent Regulation of the Follistatin Gene

Blount

Vaughan

Vale

et al. 2008

Journal of Biological Chemistry

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Activins are members of the evolutionarily conserved transforming growth factor-␤ (TGF-␤) 3 superfamily of factors implicated in the control of a wide array of cellular processes of embryonic and adult tissues (1-3). Activin A and activin B are homodimers of inhibin ␤A and ␤B subunits, respectively, with established roles in the control of many cell types including those throughout the reproductive axis (1, 4 -6). The inhibin ␤A and ␤B subunits are both expressed in the pituitary and activin B arising from gonadotropes is a critical component of the network of autocrine or paracrine factors of this tissue (7). By exerting control on FSH␤ expression, activin B is hypothesized to locally provide a positive signal for the differential production of FSH over LH and to thereby facilitate the cyclic fluctuations of these hormones during the estrus cycle (7,8). The actions of activins are strictly controlled by the concerted actions and the preferential usage of several extracellular modulators (9, 10). Of these, inhibin and follistatin have established roles in the regulation of pituitary gonadotropes (7).Follistatins are cysteine-rich glycoproteins that bind and bioneutralize activin with high affinity at a 2:1 molar ratio (11-13). They also bind and inactivate myostatin and some bone morphogenetic proteins with varying degrees of affinity (14). Structural studies of the follistatin-activin complex have elucidated the basis for this interaction and provided clues about the determinants of follistatin binding to other ligands (15,16). The follistatin gene comprising 6 exons produces two alternatively spliced mRNA transcripts and the corresponding proteins of 315 or 288 amino acids (FS315 or FS288) (17, 18). The two isoforms, FS315 and FS288, are differentially expressed in most tissues (19,20). The FS315 form is the predominant circulating form, whereas the shorter FS288 form lacking the C-terminal tail associates tightly with heparan sulfate chains of cell surface proteoglycans and is presumed to be the form that acts locally to modulate activin signaling (2,(21)(22)(23). This local function of follistatin is, in turn, controlled by the actions of activin. In cultured rat anterior pituitary cells, activin increases steadystate follistatin mRNA and secreted protein levels (24 -28). Given that inhibin antagonizes the activin-induced rise in pituitary follistatin, this pinpoints gonadotropes as the primary targets of this action of activin (28,29).Activin signals by sequentially binding to activin-specific Type II (ActRII or ActRIIB) then Type I (ALK4) serine/threonine kinase receptors to form a multimeric complex (3,30,31). In this complex, the Type II receptors trans-phosphorylate ALK4 and allow it to transiently interact and phosphorylate the downstream signaling proteins, Smad2 and Smad3, at their C-terminal SSXS motifs (31-33). The phosphorylated forms of Smad2 and Smad3 in turn translocate to the nucleus where they bind to DNA targets and regulate transcription in association with the common Smad4/DPC4 and other p...

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Production and purification of lentiviral vectors

Cited by 881 publications

References 16 publications

STIM2 regulates AMPA receptor trafficking and plasticity at hippocampal synapses

STIM2 regulates AMPA receptor trafficking and plasticity at hippocampal synapses

Knockdown of zinc finger protein X-linked inhibits prostate cancer cell proliferation and induces apoptosis by activating caspase-3 and caspase-9

A Smad-binding Element in Intron 1 Participates in Activin-dependent Regulation of the Follistatin Gene

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