technology

Mouse Hematopoietic Stem Cell Isolation, Culture, and Transplantation

Mouse hematopoietic stem cell (HSC) transplantation is a well-established in vivo model system to study various factors affecting normal and pathologic blood cell development. Based on the currently accepted classification primitive HSCs are multipotent adult stem cells that can be divided developmentally into two different stages: long-term HSCs and short-term HSCs (1). However, bone marrow contains largely heterogeneous and functionally distinct subpopulations of hematopoietic stem and progenitor cells separated by their self-renewal ability, clone size, differentiation capacity, migration patterns, and primitiveness (2). Due to this variability the reproducible isolation of HSCs representing biologically homogenous population is a very difficult task. Therefore, the characterization of HSCs is based on the in vitro functional assays, such as methylcellulose colony forming assay, or in vivo studies like competitive repopulation unit assay and the ability to form hematopoietic chimera after transplantation.

Rat Hind Limb Injury Model

Experimental hind limb injury is widely used to study the effect of transgenes, stem cells, or pharmaceuticals on e.g. new vessel formation (1,2,3,4). The degree of the injury depends on the number and the location of the vessels ligated, therefore having an impact on the recovery process and the monitoring of the transgene response. Even though terminally differentiated myofibers are not able to proliferate the skeletal muscle has a remarkable regenerative potential, which is thought to be due to the satellite cell differentiation as response to injury (5,6). Selection of the species between mouse, rat, and rabbit depends on the purpose of the study and has an impact on the method of vessel ligation. In the current review we focus on to describe the hind limb injury execution in a rat model.

Special Focus on Lentiviral Vector Development and Applications

The recent developments of viral based vectors in design, in biosafety and in accomplishing high transfection efficiency for transgene expression into target cells makes them attractive tool for various gene transfer applications. Several kinds of viruses, including HIV derived Lentivirus vectors (LVs), non-HIV based Retroviruses, Adenoviruses, Adeno-associated viruses, Baculovirus and Herpes simplex viruses have been manipulated for use in gene transfer and gene therapy purposes. Each viral vector system is integrated with a distinctive in built properties that affect its suitability for specific gene therapy purposes. Although LVs offered many unique solutions for advanced gene therapy research and clinical applications, however, several important issues of LVs gene therapy must be overcome before it gains widespread use. Nonetheless, the number of promising gene therapy studies in progress are highly encouraging and outcome from these clinical trials will provide valuable insights particularly for the clinical suitability and safety profile of LVs. This review contain a comprehensive discussion starting with the general background of the field thereafter discussing the salient features of recent developments in viral vector system in general and with special focus on LVs.

DNA Transfection

The term transfection is commonly used to describe the process of adding DNA into cells with a view to mediating protein expression of the transgene of interest. Although DNA transfection is the most commonly adopted technique, the term can also be applied to RNA transfection, where the purpose may also be to mediate protein expression or to achieve knockdown of the transcription of a specific gene, either by RNAi, ribozyme or antisense methodologies.

Use of non-integrative lentiviral vectors for gene therapy

Human Immunodeficiency Virus (HIV)-derived lentiviral vectors provide efficient gene transfer in proliferative and quiescent cells and demonstrate stable, high-level transgene expression both in vitro and in vivo. HIV non specifically integrates its DNA into the human genome, with a preference for active genes. However, integration can be problematic because a variation in gene expression between cells, possible gene silencing, and most importantly insertional mutagenesis, that can provoke malignant transformation.

Naked DNA Liver Delivery by Hydrodynamic Injection

Gene therapy is a strategy in which nucleic acid is administered for therapeutic purposes for both inherited and acquired diseases. A number of viral and nonviral vectors have been developed to circumvent the barriers for gene delivery, but the safety concerns of viral vectors have not been solved yet. On the other hand, non viral vectors are still inefficient compared to viral vectors but they offer safety as the main advantage and moreover they can be easily formulated as medicines.

Chromatin Insulators and Prospective Application for Gene Therapy

Integrating viral vectors hold great promise as gene transfer vectors for gene therapy purposes because they allow maintaining long-term expression of the therapeutic transgene throughout cell divisions. However, many issues related to integration of the provirus remain as a substantial risk for patients. The use of chromatin insulators has been proposed as a possible solution to problems raised by the integration of the vector.

Aptamer Targeting of Osteopontin in Cancer Metastasis

In recent years a novel crop of therapies called aptamers have been derived that take advantage of small segment nucleotides that tightly bind cell surface proteins. This interaction at the extracellular level impedes the normal cascade of the receptor protein, blunting or arresting the usual chain of intracellular events. In contrast to antibody directed therapies, aptamers are able to function at very low concentrations. In addition to the robust binding capability to target cell surface proteins (as described by the dissociation constant), added benefits of aptamer therapy include exquisite target specificity and a lack of immunogenicity (1, 2).