The stability of envelope-pseudotyped lentiviral vectors

Abstract Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the ‘Reduction Ratio.’ This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector’s envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2–110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.

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The NR1 subunit of the N-methyl-d-aspartate receptor can be efficiently expressed alone in the cell surface of mammalian cells and is required for the transport of the NR2A subunit

Biochemical Journal ◽

10.1042/bj3560539 ◽

2001 ◽

Vol 356 (2) ◽

pp. 539-547 ◽

Cited By ~ 5

Author(s):

Mónica GARCÍA-GALLO ◽

Jaime RENART ◽

Margarita DÍAZ-GUERRA

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Half Life ◽

Mammalian Cells ◽

Maturation Process ◽

Aspartate Receptor ◽

Heterologous System ◽

The Stability ◽

Nr2a Subunit ◽

In Cells

We have used a heterologous system of expression of N-methyl-d-aspartate (NMDA) receptors based on the use of vaccinia virus to analyse the maturation, transport, assembly and differential expression of the NR1 and NR2A subunits of the receptors. We have demonstrated that the NR1 subunit is efficiently transported to the plasma membrane in cells expressing NR1 alone, similarly to cells producing NR1 and NR2A together. In contrast, NR2A requires NR1 expression to be located at the cell surface. The stability of both receptor subunits expressed alone is similar to that obtained in cells producing NR1 and NR2A. In pulse–chase experiments, the NR1 subunit displays a biphasic decay, with a fraction of the protein having a half-life of only 1h and the remaining presenting a turnover longer than 24h, similar to values obtained for the NR2A subunit. Our results also show a maturation process affecting the carbohydrate moiety in the NR1 subunit, such that immature NR1has a much shorter half-life than the mature form or the NR2A subunit. Finally, we show that only a fraction of mature NR1 interacts with NR2A to form multimeric functional complexes.

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Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts

Biochemical Journal ◽

10.1042/bj1960377 ◽

1981 ◽

Vol 196 (2) ◽

pp. 377-382 ◽

Cited By ~ 4

Author(s):

M Caravatti ◽

J C Perriard

Keyword(s):

Creatine Kinase ◽

Myosin Heavy Chain ◽

Cell Cultures ◽

Heavy Chain ◽

Half Life ◽

Regulatory Mechanism ◽

Total Acid ◽

Myogenic Cells ◽

Creatine Kinase Isoenzyme ◽

The Stability

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2′-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.

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Lentiviral Vector Bioprocessing

Viruses ◽

10.3390/v13020268 ◽

2021 ◽

Vol 13 (2) ◽

pp. 268

Author(s):

Christopher Perry ◽

Andrea C. M. E. Rayat

Keyword(s):

Mammalian Cells ◽

Large Scale ◽

Lentiviral Vector ◽

Lentiviral Vectors ◽

Scale Production ◽

High Concentration ◽

Monogenic Diseases ◽

Flow Filtration ◽

Cell And Gene Therapy ◽

The Individual

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

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cis—Dichlorodiammineplatinum (II) and its Effect on Microfilaments in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115596 ◽

1975 ◽

Vol 33 ◽

pp. 394-395

Author(s):

S.K. Aggarwal ◽

P. McAllister

Keyword(s):

Immune System ◽

Dimethyl Sulfoxide ◽

Cell Cultures ◽

Mammalian Cells ◽

Culture Medium ◽

Cytochalasin B ◽

Fresh Medium ◽

Sarcoma 180 ◽

Heavy Meromyosin ◽

Cells In Culture

The regression of various tumors after cis-dichlorodiammineplatinum (II) (cis-Pt (II) treatment has been suggested to be mediated through mitotic inhibition and enhancement of the immune system. Present study illustrates the possible action of cis-Pt (II) on microfilaments and their role in the prevention of cytokinesis.Sarcoma-180 ascites cells in culture were treated with cis-Pt(II) (5ppm; 2 μg/ml) for intervals of 15, 30, 60 and 120 minutes. Controls were incubated in culture medium without cis-Pt (II) . Both the treated and non-treated cultures were then glycerinated and incubated with heavy meromyosin (HMM) (Ishikawa, et al., 1969. J. Cell Biol., 43, 312). Heavy meromyosin was prepared from myosin (Lowery, et al., 1969, J. Mol. Biol., 42, 1) using trypsin as inhibitor. Cell cultures treated with cis-Pt(II) for 30 minutes were also transferred to fresh medium containing 25μg/ml cytochalasin B and 1% dimethyl sulfoxide (DMSO) for 2-24 hours.

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Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

Nuklearmedizin ◽

10.1055/s-0037-1620623 ◽

1977 ◽

Vol 16 (04) ◽

pp. 157-162 ◽

Cited By ~ 1

Author(s):

C. Schümichen ◽

B. Mackenbrock ◽

G. Hoffmann

Keyword(s):

Normal Saline ◽

Half Life ◽

Compound A ◽

Short Half Life ◽

Low Concentrations ◽

The Stability ◽

Kinetics Of ◽

In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽

10.1093/genetics/163.1.91 ◽

2003 ◽

Vol 163 (1) ◽

pp. 91-101 ◽

Cited By ~ 1

Author(s):

Erin N Asleson ◽

Dennis M Livingston

Keyword(s):

Half Life ◽

Translational Regulation ◽

Cellular Level ◽

Missense Mutations ◽

Wild Type ◽

Mutant Proteins ◽

Gal1 Promoter ◽

Rad52 Protein ◽

The Stability ◽

Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

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Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22041834 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1834

Author(s):

Tomoko Okada ◽

Toshihiko Ogura

Keyword(s):

Mammalian Cells ◽

Culture Medium ◽

Related Gene ◽

Autophagosome Formation ◽

Intact Cells ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Difference ◽

Related Proteins ◽

Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

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Photoinhibition of Electron Transport Activity of Photosystem II in Isolated Thylakoids Studied by Thermoluminescence and Delayed Luminescence

Zeitschrift für Naturforschung C ◽

10.1515/znc-1988-11-1213 ◽

1988 ◽

Vol 43 (11-12) ◽

pp. 871-876 ◽

Cited By ~ 35

Author(s):

Imre Vass ◽

Narendranath Mohanty ◽

Sándor Demeter

Keyword(s):

Electron Transport ◽

Photosystem Ii ◽

Photosystem I ◽

Half Life ◽

Delayed Luminescence ◽

Transport Activity ◽

Primary Target ◽

Electron Transport Activity ◽

The Stability ◽

Spinach Thylakoids

Abstract The effect of photoinhibition on the primary (QA) and secondary (QB) quinone acceptors of photosystem I I was investigated in isolated spinach thylakoids by the methods of thermoluminescence and delayed luminescence. The amplitudes of the Q (at about 2 °C) and B (at about 30 °C) thermoluminescence bands which are associated with the recombination of the S2QA- and S2QB charge pairs, respectively, exhibited parallel decay courses during photoinhibitory treatment. Similarly, the amplitudes of the flash-induced delayed luminescence components ascribed to the recombination of S20A and S2OB charge pairs and having half life-times of about 3 s and 30 s, respectively, declined in parallel with the amplitudes of the corresponding Q and B thermoluminescence bands. The course of inhibition of thermoluminescence and delayed luminescence intensity was parallel with that of the rate of oxygen evolution. The peak positions of the B and Q thermoluminescence bands as well as the half life-times of the corresponding delayed luminescence components were not affected by photoinhibition. These results indicate that in isolated thylakoids neither the amount nor the stability of the reduced OB acceptor is preferentially decreased by photoinhibition. We conclude that either the primary target of photodamage is located before the O b binding site in the reaction center of photosystem II or QA and OB undergo simultaneous damage.

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Tafazzins from Drosophila and mammalian cells assemble in large protein complexes with a short half-life

Mitochondrion ◽

10.1016/j.mito.2015.01.002 ◽

2015 ◽

Vol 21 ◽

pp. 27-32 ◽

Cited By ~ 8

Author(s):

Yang Xu ◽

Ashim Malhotra ◽

Steven M. Claypool ◽

Mindong Ren ◽

Michael Schlame

Keyword(s):

Half Life ◽

Mammalian Cells ◽

Protein Complexes ◽

Large Protein ◽

Short Half Life

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Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2449 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2449-2454 ◽

Cited By ~ 18

Author(s):

E R Kaufman

Keyword(s):

Syrian Hamster ◽

Mammalian Cells ◽

Culture Medium ◽

Cells In Culture ◽

Thymidine Analog ◽

High Concentrations

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.

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