Re-interpreting some common objections to three transgenic applications: GM foods,xenotransplantation and germ line gene modification (GLGM)

Concerns about safety to the individual, the wider community and the potential impact on the environment are typical consequentialist objections to transgenesis that feature prominently in public debates about its ethical acceptability. I consider some of these claims with respect to their motivation, validity and their overall influence on public policy using three well-discussed applications of transgenesis: GM foods, xenotransplantation and germ line gene modification (GLGM).     

Efficient male germ line transformation for transgenic tobacco production without selection

Microspores at late uninucleate/early binucleate stages were isolated from flower buds of tobacco (Nicotiana tabacum L.) and in vitro culture methods optimised for their maturation to fully functional viable pollen which, after application to the stigma of emasculated plants in situ, led to the generation of large numbers of seed. Efficient protocols were established for the biolistic introduction of a construct containing a reporter gene and selectable marker into these microspores and hence, after in vitro maturation and in situ fertilisation, for the generation of transgenic plants. Stable transformants of low copy number were generated by this procedure. The efficiency of transformation achieved allows the production of large numbers of transgenic plants without selection, dispensing with the requirement for a selectable marker in plant transgenesis.     

Genetic transformation of mosquitoes: a quest for malaria control

Malaria inflicts an enormous toll in human lives and this burden is increasing. Present means to fight the disease, such as drugs and insecticides, are insufficient. Moreover, an effective vaccine has not yet been developed. This review examines an alternative strategy for malaria control, namely the genetic modification of mosquitoes to make them inefficient vectors for the parasite. The article summarises progress made toward the development of transposable element vectors for germ line transformation and the search for mosquito markers of transformation. Also reviewed is the search for anti-malarial effector genes whose products can inhibit development of the parasite in the mosquito with minimal fitness burden. While much progress has been made, much work remains to be done. Future research directions are discussed.     

The Insulator Effect of the 5′HS4 Region from the β-globin Chicken Locus on the Rabbit WAP Gene Promoter Activity in Transgenic Mice

Previous studies have shown that the 5'HS4 DNaseI hypersensitive site of the chicken beta-globin locus is endowed with classic insulator activities: (i) it blocks the interaction between promoter and enhancers when it is inserted between them (ii) it confers expression of integrated foreign genes independent of their position in the chromatin. The aim of this present work was to determine whether the 5'HS4 element was able to stimulate the expression level and/or to increase the expression frequency of a luc+ reporter gene controlled by the rabbit WAP gene promoter. Two constructs with 5'HS4 insulator (p5'HS4-WAPluc) or without (pWAPluc) were introduced in mouse fertilised oocytes. All transgenic lines containing the 5'HS4 element (six lines) expressed the transgene whereas only two out of eight lines harbouring the pWAP-luc construct expressed the transgene to a significant level. Moreover, the mean level of expression was seven times higher in p5'HS4WAP-luc lines than in pWAP-luc lines. Even all these benefits on transgene expression, the 5'HS4 element did not confer a copy-dependent expression, did not decrease the ectopic expression of the reporter gene and did not decrease the variability of expression. Thus, the 5'HS4 element does not have all the properties of a perfect insulator on a construct containing the luc+ reporter gene controlled by the rabbit WAP promoter.     

Generation of Ci-Brachyury-GFP stable transgenic lines in the ascidian Ciona savignyi

We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F(0) adults tested, were found to transmit the transgene to their offspring (F(1)s) at frequencies of 42% and 23%. The transgene was further inherited by the F(2) in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F(3) and F(4) generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos.     

Enhanced transgenesis by intracytoplasmic injection of envelope-free lentivirus

We demonstrate enhanced transgenesis in mice by intracytoplasmic injection of envelope-free lentivirus. Envelope-free lentivirus carrying the green fluorescent protein (GFP) gene under the control of the ubiquitin promoter (LVU-GFP) was microinjected into the cytoplasm of mouse zygotes prior to embryo transfer. Ninety-seven percent (31/32) of the adult mice were confirmed transgenic by PCR and Southern blot analysis; all founder mice express GFP when tail snips were examined by fluorescent microscopy prior to genomic DNA extraction. Transgene insertion numbers ranging from 1 to 32 were revealed by Southern blot analysis. Germline transmission was confirmed by the presence of transgene in F1 offspring. As expected, a lower transgenic rate (2.2%; 1/46) resulted when envelope-free LVU-GFP was microinjected into the perivitelline space (PVS) because cell recognition followed by membrane fusion between the viral envelope and the target cell is prerequisite for successful infection by envelope viruses. Here we demonstrate the competence of envelope-free lentivirus in establishing stable gene integration by germline transgenesis in mice at high efficiency, by intracytoplasmic viral injection (INVI) of envelope-free lentivirus into mouse zygotes.     

Development and use of a piggyBac‐based jumpstarter system in Drosophila suzukii

Spotted wing drosophila, Drosophila suzukii, is an invasive pest that primarily attacks fresh, soft‐skinned fruit. Although others have reported successful integration of marked piggyBac elements into the D. suzukii genome, with a very respectable transgenesis rate of ～16%, here we take this work a step further by creating D. suzukii jumpstarter strains. These were generated through integration of a fluorescent‐marked Minos element carrying a heat shock protein 70‐driven piggyBac transposase gene. We demonstrate that there is a dramatic increase in transformation rates when germline transformation is performed in a transposase‐expressing background. For example, we achieved transformation rates as high as 80% when microinjecting piggyBac‐based plasmids into embryos derived from one of these D. suzukii jumpstarter strains. We also investigate the effect of insert size on transformation efficiency by testing the ability of the most efficient jumpstarter strain to catalyze integration of differently‐sized piggyBac elements. Finally, we demonstrate the ability of a jumpstarter strain to remobilize an already‐integrated piggyBac element to a new location, demonstrating that our jumpstarter strains could be used in conjunction with a piggyBac‐based donor strain for genome‐wide mutagenesis of D. suzukii.     

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.