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At least 775 records · Page 43Linked to original sources
PubMed2003

[Genetic engineering potential and perspectives in the management of critical ischemia].

The paper presents a new approach to management of lower limb critical ischemia which implements recent advances in molecular biology and genetic engineering technologies. A new original compound incorporating angiogenin gene was developed to activate neoangiogenesis processes after injection into living tissues. Experimental data evidence a potential efficacy of new method for complex management of critical ischemia.

Animals↗
PubMed2006

[Separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase from culture medium of genetic engineering Pichia pastoris].

To develop a simple, fast and highly efficient method for the separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase (NbAChE) from culture medium of genetic engineering Pichia pastoris, Q-Sepharose Fast Flow chromatographic medium was used. The chromatographic column was 20 cm x 3.5 cm i. d. The elution buffer A was 20 mmol/L NaH2PO4-Na2HPO4 (pH 8) and buffer B was 1 mol/L NaCl + 20 mmol/L NaH2PO4- Na2HPO4(pH 8). The elution gradient was nonlinear. It was firstly eluted with 10% B for 300 min, then with 30% B for 300 min, finally with 100% B for 300 min. The flow rate of mobile phase was 6 mL/min. The obtained recombinant NbAChE was proved to be homogeneous on sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular mass was estimated to be approximately 66 000, which was consistent with that reported in literature. The total activity recovery of this purification method was 52.6% and the purification factor was 3.87. The final specific activity of recombinant NbAChE was 2 837 U/mg. This chromatographic process is simple and highly efficient. It can be used to separate and purify recombinant NbAChE from culture medium of Pichia pastoris harboring NbAChE gene.

Acetylcholinesterase↗
PubMed2002

Skin genetically engineered as a bioreactor or a 'metabolic sink'.

Genetically manipulated human keratinocytes can produce and secrete medically relevant proteins to the circulation. Genetically modified skin may also function as a 'metabolic sink' detoxifying the body of metabolites which accumulate in certain metabolic diseases. At the National Institutes of Health (NIH), Bethesda, Md., a clinical trial investigating the treatment of an ocular disease using the skin as a 'metabolic sink' for ornithine accumulating in gyrate atrophy patients is being prepared. The trial will involve the transplantation of a small patch of autologous keratinocytes, transduced ex vivo, onto the thighs of patients with gyrate atrophy. We are now investigating other diseases where this technology may be applicable such as in the treatment of hyperphenylalaninemia or hypercholesterolemia.

Bioreactors↗
PubMed2001

Flexible genetic engineering using RecA protein.

With the explosion in genetic information and almost complete sequencing of the human genome, a shift in the experimental goals of molecular biologists is occurring. Instead of focusing on single genes, current attempts seek to divine the interactions of several genes and sequences. This requires increasingly complex genetic constructs and manipulations, often of very large DNA constructs, and these can be made with RecA protein-based techniques. When RecA protein combined with an oligonucleotide acts as a sequence-specific "masking tape" to block DNA from the action of DNA modifying enzymes, and can be used to direct the cleavage of DNA at single predetermined restriction endonuclease sites. This reaction is called RecA-Assisted Restriction Endonuclease (RARE) Cleavage. The reverse reaction, known as RecA-Assisted Ligation, can be used to join any two desired fragments. When one of those fragments is a vector, a desired fragment can be cloned directly without constructing a genomic library. The reagents and equipment needed are relatively inexpensive, and almost any desired genetic construct up to about 300 kb in size can be made in a straightforward manner.

Animals↗
PubMed2006

Microbial considerations in genetically engineered mouse research.

Microbial infections have long been of concern to scientists using laboratory rodents because of their potential to confound and invalidate research. With the explosion of genetically engineered mice (GEM), new concerns over the impact of microbial agents have emerged because these rodents in many cases are more susceptible to disease than their inbred or outbred counterparts. Moreover, interaction between microbe and host and the resulting manifestation of disease conceivably differ between GEM and their inbred and outbred counterparts. As a result, infections may alter the GEM phenotype and confound interpretation of results and conclusions about mutated gene function. In addition, because GEM are expensive to produce and maintain, contamination by pathogens or opportunists has severe economic consequences. This review addresses how microbial infections may influence phenotype, how immunomodulation of the host as the result of induced mutations may modify host susceptibility to microbial infections, how novel host:microbe interactions have led to the development of new animal models for disease, how phenotype changes have led to the discovery of new pathogens, and new challenges associated with prevention and control of microbial infections in GEM. Although the focus is on naturally occurring infections, extensive literature on the use of GEM in studies of microbial pathogenesis also exists, and the reader is referred to this literature if microbial infection is a suspected culprit in phenotype alteration.

Animals↗
PubMed2005

Modeling medulloblastoma with genetically engineered mice.

Medulloblastoma is a malignant tumor that arises in the cerebellum in children, presumably by transformation of granule neuron precursor cells. In vivo models of medulloblastoma in genetically engineered mice have shown that activation of signal transduction pathways that stimulate proliferation and inhibit differentiation of neural progenitor cells during cerebellar development initiate medulloblastoma formation. Activation of the Sonic hedgehog (Shh)/Patched signaling pathway in the postnatal cerebellum is sufficient to induce medulloblastoma in mice. Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway by insulin-like growth factor-II, inactivation of the p53 tumor suppressor protein, loss of DNA damage repair mechanisms, and ectopic expression of Myc oncoproteins cooperate with Shh/Patched signaling to enhance tumor formation in mice. Ectopic expression of alpha and beta interferons in the developing brain also induces Shh-mediated medulloblastoma formation, suggesting a possible role for antiviral response in the genesis of medulloblastoma. By revealing which cell signaling proteins can initiate medulloblastoma formation, mouse models have enabled investigators to identify molecular targets for designing new therapies. Small-molecule inhibitors of the Shh/Patched and PI3K pathways are potential chemotherapeutic agents for patients with medulloblastoma.

Animals↗
PubMed2006

The mighty mouse: genetically engineered mouse models in cancer drug development.

Deficiencies in the standard preclinical methods for evaluating potential anticancer drugs,such as xenograft mouse models, have been highlighted as a key obstacle in the translation of the major advances in basic cancer research into meaningful clinical benefits. In this article, we discuss the established uses and limitations of xenograft mouse models for cancer drug development, and then describe the opportunities and challenges in the application of novel genetically engineered mouse models that more faithfully mimic the genetic and biological evolution of human cancers. Greater use of such models in target validation, assessment of tumour response, investigation of pharmacodynamic markers of drug action, modelling resistance and understanding toxicity has the potential to markedly improve the success of cancer drug development.

Animals↗
PubMed2002

Dendritic cells genetically engineered to simultaneously express endogenous tumor antigen and granulocyte macrophage colony-stimulating factor elicit potent therapeutic antitumor immunity.

Recently, several studies have shown that vaccine therapy using dendritic cells (DCs) genetically engineered to express a surrogate tumor antigen can effectively induce antitumor immunity. In this study, murine bone marrow DCs were adenovirally transduced with murine endogenous tumor antigen gp70 expressed in CT26 cells and granulocyte macrophage colony-stimulating factor (GM-CSF), and we examined whether antigen-specific CTL responses and therapeutic immunity could be induced in mice immunized with those genetically modified DCs. The cytotoxic activity against CT26 in mice immunized with gp70-transduced DCs was significantly higher than that in control (P < 0.01) and was enhanced by GM-CSF-cotransduction (P < 0.001). GM-CSF gene transfer into DCs expressing tumor-associated antigen enhances CC chemokine receptor 7 expression on DCs, leading to improved migratory capacity of DCs to draining lymph nodes. Consequently, an effective antitumor immune response would be induced. Vaccination using gp70-transduced DCs provided remarkable therapeutic efficacy in s.c. models. Moreover, it could be sufficiently augmented by GM-CSF-cotransduction of DCs. These results support that vaccination therapy using DCs simultaneously transduced with tumor-associated antigen can elicit potent CTL response, and GM-CSF-cotransduction of DCs could optimize therapeutic response. Further investigation is needed to optimize this vaccine therapy to achieve the obvious benefit in clinical application.

Adenoviridae↗
PubMedDate not supplied

Evaluation of an intrathecal immune response in amyotrophic lateral sclerosis patients implanted with encapsulated genetically engineered xenogeneic cells.

A phase I/II clinical trial has been performed in 12 amyotrophic lateral sclerosis (ALS) patients to evaluate the safety and tolerability of intrathecal implants of encapsulated genetically engineered baby hamster kidney (BHK) cells releasing human ciliary neurotrophic factor (CNTF). These patients have been assessed for a possible intrathecal or systemic immune response against the implanted xenogeneic cells. Hundreds of pg CNTF/ml could be detected for several weeks in the cerebrospinal fluid (CSF) of 9 out of 12 patients, in 2 patients up to 20 weeks after capsule implantation. Slightly elevated leukocyte counts were observed in 6 patients. Clear evidence for a delayed humoral immune response was found in the CSF of only 3 patients out of 12 (patients #4, #6, and #10). Characterization of the antigen(s) recognized by the antibodies present in these CSF samples allowed to identify bovine fetuin as the main antigenic component. The defined medium used for maintaining the capsules in vitro before implantation contains bovine fetuin. Fetuin may therefore still be adsorbed to the surface of the cells and/or the polymer membrane, or be present in the medium surrounding the encapsulated cells at the time of implantation. Because of the insufficient availability of CSF samples, as well as the relatively poor sensitivity of the assays used, a weak humoral immune response against components of the implanted cells themselves cannot be excluded. However, the present study demonstrates that encapsulated xenogeneic cells implanted intrathecally can survive for up to 20 weeks in the absence of immunosuppression and that neither CNTF nor the presence of antibodies against bovine fetuin elicit any adverse side effects in the implanted patients.

Adult↗
PubMedDate not supplied

Reversible hydrogels from self-assembling genetically engineered protein block copolymers.

A series of triblock protein copolymers composed of a central water-soluble polyelectrolyte segment flanked by two coiled-coil domains was synthesized by genetic engineering methods. The copolymers self-assembled into reversible hydrogels in response to changes in temperature, pH, and the presence or absence of denaturating agent (guanidine hydrochloride, GdnHCl). Hydrogel formation was concentration-dependent, and the concentration needed for hydrogel formation correlated with the oligomerization state of the coiled-coil domains in the protein copolymers. The morphology of the hydrogels, as determined by scanning electron microscopy (SEM), indicated the presence of porous interconnected networks. The thermal stabilities and self-assembling properties of the protein copolymers were successfully controlled by manipulating the amino acid sequences of the coiled-coil domains. The stimuli responsiveness and reversibility of the hydrogel self-assembly suggest that these protein copolymers may have potential in biomedical applications.

Amino Acid Sequence↗