Genetic Modification Methods Research Articles

Macronutrient innovations and their educational implications: Proteins, peptides and amino acids

Recent estimates of human requirement for indispensable amino acids have shown that the requirement for key amino acids such as lysine is twofold−threefold higher than previously thought. As a consequence recommended intakes for protein, particularly for vulnerable groups such as the elderly, need to be revised. Indispensable amino acids can also be used directly to improve amino acid supply in diets. A range of nitrogen compounds such as creatine and branched chain amino acids are currently used by groups such as athletes, although their efficacy is open to question. Peptides are natural components of the diet and some of these have been shown to have beneficial effects. The emergence of methods for genetic modification of food proteins raises possibilities of the development of novel foods for a variety of benefits including improved supply of indispensable amino acids, reduced allergenicity, or preformed antibodies to reduce risk of disease.

Ex vivo and in vivo gene transfer to the peritoneal membrane in a rat model.

The efficacy of peritoneal dialysis (PD) depends on preserving the structural integrity and dialysing capacity of the peritoneal membrane. Membrane structure and function can change on PD, resulting in decreased membrane performance and possible discontinuation of PD as a treatment modality. We hypothesized that a gene therapy strategy might be an innovative and promising approach to maintaining membrane integrity and dialysing capacity in the PD population. We have characterized two methods of genetic modification of the peritoneal membrane in a rat model to test the feasibility of this approach. In ex vivo gene transfer, mesothelial cells are isolated from the peritoneal membrane, genetically modified in culture and subsequently re-implanted back onto the peritoneal membrane of syngeneic recipients. In in vivo gene transfer, genetic modification of the membrane is accomplished in situ through adenovirus-mediated delivery of the genetic material into the peritoneal cavity. The peritoneal membrane can be genetically modified to produce factors that may be of therapeutic value in maintaining the fibrinolytic balance in the peritoneal cavity, moderating peritoneal inflammation, and studying the development of peritoneal fibrosis. These models provide a basis for studying the contribution of specific molecules to peritoneal membrane physiology. Optimally, they will be paired with other PD-relevant model systems to understand peritoneal physiology, identify ways to prevent membrane damage, and maintain dialysing performance. While gene transfer can be used as a tool to understand the individual roles of factors or pathways in peritoneal membrane physiology, it can also be developed as a therapy platform for improving membrane characteristics and enhancing the therapy of peritoneal dialysis.

Germ cell transplantation from large domestic animals into mouse testes.

Donor-derived spermatogenesis after spermatogonial transplantation to recipient animals could serve as a novel approach to manipulate the male germ line in species where current methods of genetic modification are still inefficient. The objective of the present study was to investigate germ cell transplantation from boars, bulls, and stallions, which are economically important domestic animals, to mouse recipients. Donor testis cells (fresh, cryopreserved, or cultured for 1 month) were transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. Recipient testes were analyzed from 1 to > 12 months after transplantation for the presence of donor germ cells by donor-specific immunohistochemistry. Donor cells were present in most recipient testes with species-dependent differences in pattern and extent of colonization. Porcine donor germ cells formed chains and networks of round cells connected by intercellular bridges but later stages of donor-derived spermatogenesis were not observed. Transplanted bovine testis cells initially appeared similar but then developed predominantly into fibrous tissue within recipient seminiferous tubules. Few equine germ cells proliferated in mouse testes with no obvious difference between cells recovered from a scrotal or a cryptorchid donor testis. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh or cryopreserved germ cells from large animals can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion. Species-specific differences in the compatibility of large animal donors and mouse recipients were detected which cannot be predicted solely on the basis of phylogenetic distance between donor and recipient species.

Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid.

Simple, nontoxic, and pharmaceutically defined methods for genetic modification of respiratory tissues may enable development of a variety of molecular medicines. Clinical applications for such medicines include treatment of inborn errors of metabolism, interventions for asthma and iatrogenic pulmonary fibrosis, and disease prophylaxis via mucosal polynucleotide vaccination. "Free," "direct," or "naked" plasmid administration is a simple, apparently safe, and pharmaceutically defined gene delivery method. Murine, macaque, and clinical human studies have demonstrated transfection of respiratory tissues after direct application of free plasmid. The aim of this study was to develop a simple and safe alternative to respiratory tissue transduction, and specifically to provide a theoretical framework for developing a category of adjuvants, nuclease inhibitors, that augment the transfection activity of free plasmid. Plasmid employing the human CMV IE promoter/enhancer to drive expression of the Photinus pyralis luciferase reporter protein was administered intratracheally into mouse lung with or without the nuclease inhibitor aurintricarboxylic acid (ATA). Lavage samples and tissue extracts were used to demonstrate inhibition of lung nuclease activity. ATA dose escalation studies were performed using lung homogenate assays to characterize transfection. Potential toxicity was assessed histologically. The data indicate that nucleases present in respiratory fluids accelerate clearance of biologically active plasmid from lung, that intratracheal coadministration of ATA together with plasmid reduces extracellular DNA clearance, and that this treatment results in marked enhancement of reporter protein expression. The effective dose for ATA enhancement of direct lung transfection was 0.5 microg/g mouse weight, and the LD50 was approximately 6 microg/g. These findings provide a theoretical and practical foundation for further development of an alternative gene delivery system: free plasmid-based respiratory transfection technology.

The impact of hybrids between genetically modified crop plants and their related species: general considerations

AbstractFactors influencing the fate and impact of hybrids between crop plants and their related species operate from the early zygote, through to plant establishment in different habitats, to their ability to form self‐sustaining populations. Many of the classes of genes being introduced by modern methods of genetic modification are similar to those manipulated by conventional plant breeding. In assessing the impact of transgenes in hybrids between crops and related species, therefore, it is important to be informed about the consequences of hybridization between conventionally bred varieties and their relatives. Some transgenes will have novel effects (e.g. production of pharmaceutical substances or certain fatty acids) on plants, and are likely to need specific assessment studies to determine their impact on hybrids. This will be particularly important if there is the possibility of these transgenes becoming established in wild populations. Some recommendations for further research are outlined.

The Experimental and Commercial Release of Transgenic Crop Plants

AbstractWith advances in recombinant DNA methods and transformation procedures, it is possible to transfer genes into crop plants from unrelated plants, microbes and animals. Many of the modifications being carried out, or envisaged, are for disease and pest resistance, product quality and tolerance to environmental stress, but there are additional opportunities to modify crops to give specialized products for industrial or pharmaceutical use. Some of the characteristics of transgenic plants are considered, including: transgene copy number, position, expression, stability, pleiotropy, selectable marker genes and somaclonal variation. There have been several hundreds of field trials with transgenic plants, and the first transgenic varieties are likely to be approved for commercial production in 1993. Before releasing transgenic plants, it is necessary to carry out a risk assessment to determine whether the transgenic variety will behave differently from a conventionally bred variety. Assessment procedures are being harmonized internationally by various organizations. There is a growing commitment to apply these genetic modification methods to crops in developing countries, as genes relevant to their crops and environments become available.

The safety of foods developed by biotechnology.

Recombinant DNA techniques are now being used to develop new plant varieties that will be sources of foods, such as fruits, vegetables, grains, and their by-products. These techniques enable developers to make specific genetic modifications in plants, including modifications that introduce substances into plants that could not be introduced by traditional methods. To ensure the safety of the resulting foods and to foster innovation, the FDA is taking the initiative, before foods from such plants are ready to enter the market, to see that there is an agreed upon scientific basis to evaluate the safety of whole foods and animal feeds derived from new plant varieties. Here, the authors summarize their regulatory framework and their approach to safety assessment and discuss its scientific basis. Their safety assessment approach addresses new varieties of food crops developed by both traditional and newer methods of genetic modification and provides guidance on how safety issues should be addressed.

Chapter 3: Methods of genetic modification and their use

Chapter 3: Methods of genetic modification and their use

Utility of isoelectric focusing techniques for detection of induced in vivo mammalian microlesions

We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.