遗传学实验技术

Two-dimensional (2D) electrophoretic fractionation of digested DNA has been useful in analyzing and comparing the genomic structure of viruses and prokaryotic organisms (1–4). For eukaryotes, it has been used to examine the genomic distribution of multicopy gene families (5), to dete ...

Central to the regulation of transcription of eukaryotic genes is the interaction of specific DNA binding proteins with promoter and enhancer elements. The molecular cloning of such DNA binding factors is an important step toward understanding this process and a number of strategies ha ...

The 6xHis/Ni-NTA system is a fast and versatile tool for the affinity purification of recombinant proteins and antigenic peptides. It is based on the high-affinity binding of six consecutive histidine residues (the 6xHis tag) to immobilized nickel ions, giving a highly selective interac ...

Although a member of the lentivirus group, the equine infectious anemia virus (EIAV) was the first nonplant virus discovered in the first decade of the 20th century (1), lentiviruses were considered as rather mysterious viruses until the isolation of the human immunodeficiency virus type 1 ( ...

Transducing lentiviral vectors that transfer the exogenous gene(s) into target cells are typically defective for viral replication because at least some of the trans-acting sequences encoding the viral proteins have been deleted. To propagate vector virus, the viral proteins are su ...

Human immunodeficiency virus (HIV)-derived lentiviral vectors can integrate into the genome of dividing and nondividing cells, in vitro as well as in vivo (reviewed in refs. 1–3). Lentiviral vectors are particularly efficient in transducing multipotent stem cells, such as hematopoi ...

Lentiviral vectors have traditionally been produced in human embryonic kidney 293T cells (1), involving transient transfection procedures that were originally established for the production of retroviral vectors based on Moloney murine leukemia (MoMLV) virus (2). Simian vir ...

The accurate quantification of functional vector particles within a given vector preparation or stock is of fundamental importance to virtually all potential gene transfer applications for any viral vector of interest. This accuracy is especially critical if anticipated clini ...

Lentiviral vectors derived from human immunodeficiency virus (HIV) have demonstrated exceptional promise as tools for gene therapy applications (1,2), but have also raised safety concerns because of potential creation of replication-competent lentivirus (RCL) by uncontro ...

Lentiviruses are members of a subgroup of enveloped Retroviridae. Lentivirus-based gene delivery vectors have gained popularity in gene therapy field because of their notable potentials in delivering and integrating transgenes into both mitotic active and inactive cells in vit ...

There has been much effort in isolating hematopoietic stem cells (HSC) and some confusion within the literature on what even a HSC is, with some investigators using the term inappropriately (1). A HSC is a cell capable of both self-renewal and of deriving progeny of all hematopoietic cell lineages, t ...

It has proven notoriously difficult to express foreign genes in primary neuronal cells in vitro. Some success has been achieved with lipid-mediated plasmid DNA transfection (1), transduction via mechanical methods (e.g., gene gun) (2,3), and infection with a variety of neurotropic viral v ...

Adult bone marrow contains both hematopoietic and nonhematopoietic stem cells, also termed mesenchymal stem cells (MSCs), or mesenchymal progenitor cells. MSCs are cells capable of differentiating into cells of the mesenchymal lineage (1–3). These cells were previously referred ...

Conventional ways of introducing genes into cells, such as calcium phosphate transfections and liposome or lipofectamine-mediated methods, work poorly in cardiomyocytes. They can be useful for gene reporter studies, but are of little value when a gene needs to be introduced into a large nu ...

The ability of lentivirus vectors to stably transduce postmitotic cells by genomic integration and early evidence of a capacity for retrograde axoplasmic transport, depending on the envelope pseudotype, underpin the immense potential of this vector system for neuronal transduc ...

Cystic fibrosis (CF) is a common inherited disorder affecting a variety of epithelial tissues. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that lead to abnormal secretions, recurrent infection and inflammation, bronc ...

A mammalian cornea can be maintained in vitro for days (1,2), allowing ex vivo transduction of this tissue prior to transplantation (3,4). Ex vivo transduction of the cornea is useful for studying the efficacy of gene products on inhibition of corneal neovascularization (4), amelioration of co ...

Mutations within genes that are specifically expressed within the retina and/or the retinal pigment epithelium (RPE) are associated with the majority of inherited retinal degenerations (1). Retinal degenerative disorders are highly heterogeneous, both genetically and phen ...

The manipulation of gene expression in primary lymphocytes has been shown to have potential therapeutic applications for immunodeficiencies (1–3), and cancer immunotherapy (4,5). Efficient gene transfer is an absolute prerequisite for developing successful clinical gene th ...

Dendritic cells (DCs) are a heterogeneous population of bone marrow-derived antigen-presenting cells (APCs) that populate various lymphoid and nonlymphoid tissues, including the skin (Langerhans cells), lymph nodes (interdigitating and follicular DCs), spleen, and thymus. ...