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The author's own data and those available in literature concerning the mutagenic action of viruses and recombinant DNA molecules on mammalian cells permit drawing attention to the possible hazards of genetic engineering which might affect the genome of mammals, including a man.
More than 2 decades have past since the first transgenic mouse was created. In this review we will focus on recent technologies in the field of mouse genetic engineering and on its application to bioindustry. The most important milestone in this field was the invention of the gene targeting technique. Recent improvement of this technique to conditional gene targeting together with the Cre/IoxP system further encouraged the generation of mutant mice. It is obvious that there will be a great demand for some mutant mice as experimental models of human disease or as resources of experimental material for basis biological research. Thus, creation of such mice would be a rewarding challenge for a biological enterprise.
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The influence of environmental conditions such as pH, temperature, and ionic strength on the equilibrium swelling ratio of physically crosslinked networks of a genetically engineered silk-elastinlike protein-based copolymer (SELP) with an amino acid repeat sequence of [(GVGVP)4GKGVP(GVGVP)3(GAGAGS)4]12 was investigated. The effects of gelation cure time and initial polymer concentration on the equilibrium swelling ratio and soluble fraction of the hydrogels were also studied. It was found that the soluble fraction linearly correlated with the initial polymer concentration at higher gelation times. Soluble fraction results suggest that final hydrogel water content may be controlled by both initial polymer concentration and gelation time. Equilibrium swelling studies demonstrated that these hydrogels are relatively insensitive to environmental changes such as pH, temperature, and ionic strength. Over the concentration range studied, it was found that an increase in gelation time at 37 degrees C resulted in lower hydrogel weight equilibrium swelling ratios, which corresponds to less soluble polymer released post-gelation. Together, these results have implications for the controlled delivery of bioactive agents from silk-elastinlike hydrogels.
Since endothelial cells (EC) are the major target cells during hyperacute rejection and are likely in delayed graft rejection, most of the genetic engineering of the xenotransplant donor is aimed at modifying their properties. Among the various strategies that are reviewed are the genotypic or phenotypic knockout of the alpha 1,3Gal antigen, which is a major target of xenoantibodies and is also probably involved in innate cellular response. In addition, the success of the transgeny of complement regulatory proteins is well established. In vitro data from analyses of the mechanisms of endothelial cell activation also suggest that other molecules could be used to regulate apoptosis or thrombotic microenvironment or to minimize recipient T-cell activation by inhibiting costimulatory proteins such as CD40 or B7. Alternative to usual knockout techniques (thus far not available in pigs, where no ES cells have been derived) will be presented.
The construction, synthesis and expression of a genetically engineered bifunctional antibody/cytokine fusion protein is described. To target IFN-tau to tumor cells, recombinant antibody techniques were used to construct a RM4/IFN-tau fusion protein containing the chimeric anti-tumor F(ab')2 (RM4) and the IFN-tau moiety. The recombinant cDNA of IFN-tau was linked to 3 prime end of the chimeric heavy-chain gene fragment (M4) containing the VH, the CH1 and the hinge region to form the fused heavy-chain gene fragment M4-IFN-tau. Transfection of the M4-IFN-tau gene fragment into a myeloma derived cell line VKCK which produced the chimeric light-chain of the same antibody, allowed the transfectant secreting the bifunctional fusion protein RM4/IFN-tau. The RM4/IFN-tau was purified by the affinity chromatography. Our data showed that the RM4/IFN-tau retained the TAG72 antigen-binding reactivity as well as the IFN-tau activity as measured in ELISA, FACS analysis of cell-surface TAG72 expression, immunohistochemical study, and up-regulation of cell-surface expression of CEA, HLA class I and class II antigens. Therefore, the bifunctional fusion protein RM4/IFN-tau may prove to be useful in targeting biological effects of the IFN-tau to tumor cells and in this way to stimulate the immune destruction of tumor cells.
A bacterial sensing system that responds selectively to antimonite and arsenite has been investigated. The bacteria used in these studies have been genetically engineered to produce the enzyme beta-galactosidase in response to these ions. This is accomplished by using a plasmid that incorporates the gene for beta-galactosidase (reporter gene) under the control of the promoter of the ars operon. This plasmid also encodes for the ArsR protein, a regulatory protein of the ars operon, which, in the absence of antimonite or arsenite, restricts the expression of beta-galactosidase. In the presence of antimonite or arsenite the ArsR protein is released from the operator/ promoter region of the ars operon and beta-galactosidase is expressed. The activity of this enzyme was monitored electrochemically using p-aminophenyl beta-D-galactopyranoside as the substrate. The bacterial sensing system responds selectively to arsenite and antimonite (and to a lesser extent arsenate) and shows no significant response to phosphate, sulfate, nitrate, and carbonate.
Thermally associating polymers, including gelatin, cellulose ethers (e.g., Methocels and poloxamers (e.g., Pluronics) have a long history of use in pharmacy. Over the past 20 years, significant advances in genetic engineering and the understanding of protein secondary and tertiary structures have been made. This has led to the development of a variety of polypeptides that do not occur naturally but can be expressed in recombinant cells and have useful properties that lend themselves to novel applications where current materials cannot perform. The most intensively studied motifs are derived from the consensus repeats of elastin and silk, as well as coiled-coil helices. Many of these designed polypeptides or 'artificial proteins' are thermally associating materials. This property can be exploited to develop solid dosage forms, injectable drug delivery systems, micro- or nanoparticle drug carriers, triggered or targeted release systems, or as a means of simplifying the purification process and thus reducing costs of production of these materials. This review focuses on the development and characterization of this novel class of biomaterials and examines their potential for pharmaceutical applications.
Recently, a C to T transition mutation in exon 8 of the p53 gene has been identified in a subculture of the genetically engineered human lymphoblastoid cell line AHH-1 and this mutation was proposed to cause a loss of function of the p53 suppressor protein and may limit the use of this cell culture in genotoxicology test assays. This led us to investigate early passage cultures of AHH-1 and its derivative MCL-5 to determine the distribution of the mutation. In order to characterise the presence of mutations at the p53 locus, exon 8 was analysed using restriction enzyme analysis and automated sequencing to locate possible changes of sequence. Mutations were identified at codon 282, and treatment with the Msp1 restriction enzyme led to incomplete digestion suggesting the presence of heterozygosity at the site which was confirmed by sequencing. Our results indicate that the p53 gene is heterozygous at the interface between the codons 281 and 282 in both AHH-1 and MCL-5. An Annexin V labeling study was carried out and both AHH-1 and MCL-5 cell lines were shown to undergo DNA damage induced cell death after a 1-h exposure to MNNG.
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The Pathological Classification of Prostate Lesions in Genetically Engineered Mice (GEM) is the result of a directive from the National Cancer Institute Mouse Models of Human Cancer Consortium Prostate Steering Committee to provide a hierarchical taxonomy of disorders of the mouse prostate to facilitate classification of existing and newly created mouse models and the translation to human prostate pathology. The proposed Bar Harbor Classification system is the culmination of three meetings and workshops attended by various members of the Prostate Pathology Committee of the Mouse Models of Human Cancer Consortium. A 2-day Pathology Workshop was held at The Jackson Laboratory in Bar Harbor, Maine, in October 2001, in which study sets of 93 slides from 22 GEM models were provided to individual panel members. The comparison of mouse and human prostate anatomy and disease demonstrates significant differences and considerable similarities that bear on the interpretation of the origin and natural history of their diseases. The recommended classification of mouse prostate pathology is hierarchical, and includes developmental, inflammatory, benign proliferative, and neoplastic disorders. Among the neoplastic disorders, preinvasive, microinvasive, and poorly differentiated neoplasms received the most attention. Specific criteria were recommended and will be discussed. Transitions between neoplastic states were of particular concern. Preinvasive neoplasias of the mouse prostate were recognized as focal, atypical, and progressive lesions. These lesions were designated as mouse prostatic intraepithelial neoplasia (mPIN). Some atypical lesions were identified in mouse models without evidence of progression to malignancy. The panel recommended that mPIN lesions not be given histological grades, but that mPIN be further classified as to the absence or presence of documented associated progression to invasive carcinoma. Criteria for recognizing microinvasion, for classification of invasive gland-forming adenocarcinomas, and for characterizing poorly differentiated tumors, including neuroendocrine carcinomas, were developed and are discussed. The uniform application of defined terminology is essential for correlating results between different laboratories and models. It is recommended that investigators use the Bar Harbor Classification system when characterizing new GEM models or when conducting experimental interventions that may alter the phenotype or natural history of lesion progression in existing models.
A number of recent technological developments have greatly facilitated the genetic engineering of immunoglobulins. The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunised cells. The conversion of the rodent antibody into a humanized version is now well established. To develop these antibodies for clinical use has required the development of high level expression systems. For the expression of large multimeric glycoproteins, mammalian cell systems generally provide the highest levels of secreted product and therefore are the methods of choice for producing whole recombinant antibodies. Novel antigen-binding units have been developed by joining the two variable domains of an antibody into single-chain polypeptides. Such fragments can be produced in high yield by secretion from E. coli raising the prospect of bulk preparation of these antibody fragments for the development of low-cost immunopurification and assay reagents. Finally, the ability to screen for antigen binding by displaying immunoglobulin variable regions on the surface of filamentous bacteriaphages has opened up the possibility of bypassing the immune system to generate novel antibody specificities in vitro.
OBJECTIVE: To develop genetically engineered retinal pigment epithelial (RPE) cells which are able to express bioactive nerve growth factor (NGF) steadily and efficiently and to combine gene therapy with retinal transplantation techniques to treat retinal degeneration. METHODS: Replication-deficient retrovirus-plasmid recombinant pLXSN-NGF was constructed via subclone and clone, and then packaged into pT67 cells to obtain virion containing a secretable form of NGF gene. We evaluated titer of the retroviral recombinant with NIH 3T3 cells and then transduced NGF gene into cultured RPE cells by the retroviral recombinant. We also analyzed the expression of NGF gene in transduced and untransduced RPE cells via reverse transcription (RT)-PCR and Western blot. The bioactivity of expressed NGF were evaluated by stimulating PC 12 cells with conditioned medium from transduced and untransduced RPE cells. RESULTS: The titer of replication-deficient retroviral recombinant reached 1.2 x 10(7) cfu/ml. NGF protein and mRNA were detected in the conditioned medium from transduced RPE cells, but not from the normal RPE cells. Marked neurite process outgrowth from PC 12 cells was observed after being stimulated with the conditioned medium from transduced RPE cells. CONCLUSIONS: RPE cells are able to be transduced by replication-deficient retrovirus with high efficiency, and secret bioactive NGF at a high level, which might be an ideal candidate for engineered cells.
This review describes work aimed at the improvement of the nutritive value of grain and forage legumes using gene transfer techniques. Two traits which are amenable to manipulation by genetic engineering have been identified. These are plant protein quality and lignin content. In order to increase the quality of protein provided by the legume grains peas and lupins, we are attempting to introduce into these species chimeric genes encoding a sunflower seed protein rich in the sulphur-containing amino acids methionine and cysteine. These genes are designed to be expressed only in developing seeds of transgenic host plants. Chimeric genes incorporating a similar protein-coding region, but different transcriptional controls, are being introduced into the forage legumes lucerne and subterranean clover. In this case the genes are highly expressed in the leaves of transformed plants, and modifications have been made to the sunflower seed protein-coding sequences in order to increase the stability of the resultant protein in leaf tissue. Another approach to increasing plant nutritive value is represented by attempts to reduce the content of indigestible lignin in lucerne.