Abstract:
:Gene therapy vectors based on murine retroviruses have now been in clinical trials for over 20 years. During that time, a variety of novel vector pseudotypes were developed in an effort to improve gene transfer. Lentiviral vectors are now in clinical trials and a similar evolution of vector technology is anticipated. These modifications present challenges for those producing large-scale clinical materials. This chapter discusses approaches to process development for novel lentiviral vectors, highlight considerations, and methods to be incorporated into the development schema.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Leath A,Cornetta Kdoi
10.1016/B978-0-12-386509-0.00005-3subject
Has Abstractpub_date
2012-01-01 00:00:00pages
89-108eissn
0076-6879issn
1557-7988pii
B978-0-12-386509-0.00005-3journal_volume
507pub_type
杂志文章abstract::High content, phenotypic screens offer a powerful approach to systems biology at the cellular level. The approach employs cells carrying fluorescently labeled molecules or organelles in 384- or 1536-well microplates, and an automated confocal screening microscope for capturing images from each well. Although some spec...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.09.021
更新日期:2018-01-01 00:00:00
abstract::Here we describe an optimized recombineering protocol for the creation of conditional alleles. Recombineering is a DNA engineering methodology based on homologous recombination in Escherichia coli. It presents a number of advantages over conventional engineering methods, including the ability to place target sites for...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)77008-7
更新日期:2010-01-01 00:00:00
abstract::Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2014.12.030
更新日期:2015-01-01 00:00:00
abstract::Reference standard materials (RSMs) exist for a variety of biologics including vaccines but are not readily available for gene therapy vectors. To date, a recombinant adeno-associated virus serotype 2 RSM (rAAV2 RSM) has been produced and characterized and was made available to the scientific community in 2010. In add...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-386509-0.00015-6
更新日期:2012-01-01 00:00:00
abstract::A neuron is able to seamlessly respond to a number of signals, in a timely and specific manner. This process, of integrating multiple inputs, relays on the orchestration of intracellular events by signaling networks. The inherent complexity of signaling networks has made computational modeling a useful approach to und...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-388448-0.00014-0
更新日期:2012-01-01 00:00:00
abstract::Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycos...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385928-0.00010-9
更新日期:2011-01-01 00:00:00
abstract::Plots of free energy versus temperature are commonly called stability curves or Gibbs-Helmholtz curves, and they have proven to be extremely useful in protein folding and ligand-binding studies. Curvature in a Gibbs-Helmholtz or stability plot is indicative of a heat capacity change, and some of their primary uses in ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381268-1.00009-4
更新日期:2011-01-01 00:00:00
abstract::Specific recognition of mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Structural determination of the eIF4E-cap complexes, as well as complexes of eIF4E with other proteins regulating its activity, requires complementary experiments that allow for energetic an...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)30009-8
更新日期:2007-01-01 00:00:00
abstract::Contemporary high-throughput sequencing methods, notably RNA-Seq, permit the systematic identification and characterization of transcripts whose levels change significantly in response to altered biological states. We have described methods for the application of this methodology to a definition of the transcripts reg...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.09.005
更新日期:2018-01-01 00:00:00
abstract::Differential scanning calorimetry (DSC) detects protein thermal unfolding by directly measuring the heat absorbed. Simple DSC experiments that require relatively small amounts of pure material can provide a wealth of information related to structure, especially with respect to domain architecture, without the need for...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.014
更新日期:2016-01-01 00:00:00
abstract::IF-liposomes are formed by a unique process that involves fusing small liposomes into interdigitated lipid sheets, using either ethanol or hydrostatic pressure. The interdigitation-fusion method requires liposome formulations with lipids that form the L beta I phase. Preparing ethanol-induced IF-liposomes is simple an...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(03)67007-2
更新日期:2003-01-01 00:00:00
abstract::Model building studies may be used to supplement structurally low resolution experimental data with detailed three-dimensional hypothetical atomic models. Because of the strong relation between structure and function in biological molecules such models may give a consistent, integral view of a wealth of experimental d...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/0076-6879(92)11024-d
更新日期:1992-01-01 00:00:00
abstract::In order to produce a full quantitative description of yeast metabolism, a number of kinetic parameters of enzymes that are important for energy metabolism must be determined experimentally. We aim to determine the prospective in vivo kinetic properties of a range of yeast-purified isoenzymes that are important in ene...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385118-5.00012-8
更新日期:2011-01-01 00:00:00
abstract::Extracellular signals dictate the biological processes of neural stem cells (NSCs) both in vivo and in vitro. The intracellular response elicited by these signals is dependent on the context in which the signal is received, which in turn is decided by previous and concurrent signals impinging on the cell. A synthesis ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)19002-3
更新日期:2006-01-01 00:00:00
abstract::The characterization of microbial adhesins is greatly facilitated by the use of synthetic peptides. Synthetic peptides can be used to identify specific antigenic epitopes, to delineate receptor-binding domain of adhesins, and to facilitate the characterization of the adhesin, and they allow for a direct examination of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(95)53013-4
更新日期:1995-01-01 00:00:00
abstract::The genome integrity of living organisms is constantly threatened by endogenous cellular metabolic processes and environmental agents. To quantify these low, physiologically relevant levels of DNA damage, a single-cell gel electrophoresis (comet) combined with strand-specific fluorescence in situ hybridization (FISH)-...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.03.023
更新日期:2017-01-01 00:00:00
abstract::The remarkable success of cancer immunotherapies, especially the checkpoint blocking antibodies, in a subset of patients has reinvigorated the study of tumor-immune crosstalk and its role in heterogeneity of response. High-throughput sequencing and imaging technologies can help recapitulate various aspects of the tumo...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.01.001
更新日期:2020-01-01 00:00:00
abstract::The epitope tagging approach offers advantages of economy, universality, and precision over the use of antibodies raised directly against a protein of interest. The latter strategy promises a potentially greater diversity of reagents and obviates the need to modify the protein, but it may not yield sufficiently high-a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(91)94038-e
更新日期:1991-01-01 00:00:00
abstract::Sequence analysis of synthetic peptides using Edman chemistry can be very useful for the elucidation of certain types of synthetic problems, such as residue deletions and the presence of common stable derivatives, and for following the progress of the synthesis itself. However, it can also be a relatively poor techniq...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(97)89056-8
更新日期:1997-01-01 00:00:00
abstract::When isolating ribonucleoprotein (RNP) complexes by an affinity selection approach, tagging the RNA component can prove to be strategically important. This is especially true for purifying single types of snoRNPs, because in most cases the snoRNA is thought to be the only unique component. Here, we present a general s...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)25014-1
更新日期:2007-01-01 00:00:00
abstract::Lysosomes are specialized organelles for protein recycling and as such are involved in the terminal steps of autophagy. However, it has become evident that lysosomes also play an important role in the progression of apoptosis. This latter function seems to be dependent on lysosomal proteases, which need to be released...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(08)01409-2
更新日期:2008-01-01 00:00:00
abstract::Atomic force microscopy (AFM) enables direct visualization of single-protein molecules in liquids at submolecular resolution. High-speed AFM further makes it possible to visualize dynamic biomolecular processes at subsecond resolution. However, dynamic imaging of biomolecular processes imposes various requirements on ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)75020-5
更新日期:2010-01-01 00:00:00
abstract::Guanine nucleotide exchange factors (GEFs) are essential regulators of the spatiotemporal conditions of small GTP-binding protein (SMG) activation. Their cellular activities combine the biochemical stimulation of GDP/GTP exchange, which leads to the active conformation of the SMG, to the detection of upstream signals ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)38004-X
更新日期:2008-01-01 00:00:00
abstract::Crystallographic studies on flavodiiron proteins (FDPs) have revealed that the common sequence core ( approximately 400 residues) that defines this protein family comprises two structural domains. The N-terminal domain (of approximately 250 residues) displays a metallo-beta-lactamase-like-fold, being indeed structural...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)37001-8
更新日期:2008-01-01 00:00:00
abstract::Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid-protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of thi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)69009-1
更新日期:2009-01-01 00:00:00
abstract::Hypoxia and inflammation are coincidental events in an array of diseased tissues, including chronically inflamed sites (e.g., inflammatory bowel disease, rheumatoid arthritis), growing tumors, myocardial infarcts, atherosclerotic plaques, healing wounds, and sites of bacterial infection (Murdoch et al., 2005). An unde...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)35025-8
更新日期:2007-01-01 00:00:00
abstract::Cell wall precursors that have been modified at their peptide moiety were incorporated into the living bacterial cell wall. Using chemically synthesized bacterial cell wall precursors, a variety of compounds could be attached to the bacterial surface. Escherichia coli took the modified precursors into the cell wall af...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(03)01019-x
更新日期:2003-01-01 00:00:00
abstract::The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availabili...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(89)78005-8
更新日期:1989-01-01 00:00:00
abstract::Adenoviral vectors expressing vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A(164)) offer a powerful method for elucidating the mechanisms of pathological angiogenesis and lymphangiogenesis and for evaluating the effectiveness of pro- and anti-angiogenesis therapies. When injected int...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02803-6
更新日期:2008-01-01 00:00:00
abstract::Receptor ensemble docking (RED) is an effective strategy to account for receptor flexibility in the course of a docking-based virtual screening campaign. Such an approach can be applied when multiple crystal structures of a receptor have been solved, but it can also be applied when only a single crystal structure is a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-407865-9.00014-5
更新日期:2013-01-01 00:00:00