COMMENTARY ON ZOHAR'S “PROSPECTS FOR‘GENETIC THERAPY’- CAN A PERSON BENEFIT FROM BEING ALTERED?”

In his paper on the effects of Prenatal Genetic Intervention (PGI) on personal identity, Noam Zohar comes to a conclusion about genetic makeup and the uses of gene therapy quite different from the one I reach in another piece in this issue. Zohar's argument rests on the contention that personal identity changes with alteration of the genome, following what I have identified as the "constitutive" view. To see that this is the pillar supporting the weight of his argument, consider the following. Questions of identity aside, how can it be that altering the genome of children suffering from Lesch-Nyhan syndrome or Tay-Sachs disease so that they now produce the enzyme that they formerly lacked does not benefit them? Clearly, if their identities were not changed, such individuals would in fact realize great benefit from PGI, since the devastating bad effects of the genetic flaw would be avoided. Such a change would certainly make the altered individuals better off, that is, it would benefit them. On this, Zohar and I do not disagree. Persistence of identity through such genetic change is the sticking point.     

Conditional gene knockout using cre recombinase

Cre recombinase has become an important instrument for achieving precise genetic manipulation in mice. Many of these desired genetic manipulations rely on Cre’s ability to direct spatially and temporally specified excision of a predesignated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site. Success in achieving such conditional mutagenesis in mice depends both on the careful design of conditional alleles and on reliable detection of cre gene expression. These procedures include PCR, immunohistochemistry and the use of a recombination-proficient GFP-tagged Cre protein.     

GENE DOPING IN SPORT – PERSPECTIVES AND RISKS

In the past few years considerable progress regarding the knowledge of the human genome map has been achieved. As a result, attempts to use gene therapy in patients’ management are more and more often undertaken. The aim of gene therapy is to replace defective genes in vivo and/or to promote the long-term endogenous synthesis of deficient protein. In vitro studies improve the production of human recombinant proteins, such as insulin (INS), growth hormone (GH), insulin-like growth factor-1 (IGF-1) and erythropoietin (EPO), which could have therapeutic application. Unfortunately, genetic methods developed for therapeutic purposes are increasingly being used in competitive sports. Some new substances (e.g., antibodies against myostatin or myostatin blockers) might be used in gene doping in athletes. The use of these substances may cause an increase of body weight and muscle mass and a significant improvement of muscle strength. Although it is proven that uncontrolled manipulation of genetic material and/or the introduction of recombinant proteins may be associated with health risks, athletes are increasingly turning to banned gene doping. At the same time, anti-doping research is undertaken in many laboratories around the world to try to develop and refine ever newer techniques for gene doping detection in sport. Thanks to the World Anti-Doping Agency (WADA) and other sports organizations there is a hope for real protection of athletes from adverse health effects of gene doping, which at the same time gives a chance to sustain the idea of fair play in sport.     

Experimental evidence of cost of lactation in a low risk environment for a long-lived mammal

In a previous experiment we have documented that organisms adopt a risk-sensitive reproductive allocation when summer reproductive investment competes with survival in the coming winter ( Bårdsen et al. 2008 ). This tradeoff is present through autumn female body mass, which acts as an insurance against unpredictable winter environmental conditions. We tested this hypothesis experimentally on female reindeer experiencing stable and benign winter feeding conditions. Additional supplementary feeding and removal of newborns represented two sets of experimental manipulations. Females in the supplementary feeding group increased more in winter body mass relative to control females. This manipulation, however, did not have any effect on summer body mass development for neither females nor offspring, but we found a positive effect of feeding on offspring birth mass for smaller females. In contrast, offspring removal did have a positive effect on summer body mass development as females in this group were larger in the autumn relative to control females. In essence, we documented two immediate effects as: (1) supplementary feeding did have a positive effect on spring body mass for smaller females; and (2) offspring removal did increase the female summer somatic growth as this had a positive effect on female autumn body mass. Additionally, we tested for lagged effects, but we could not document any biologically significant effects of neither manipulation in the coming spring. The fact that we only found rather weak effects of both manipulations was as expected for risk sensitive individuals experiencing benign environmental conditions over many years.     

Cre recombinase induces DNA damage and tetraploidy in the absence of LoxP sites

The spatiotemporal manipulations of gene expression by the Cre recombinase (Cre) of bacteriophage P1 has become an essential asset to understanding mammalian genetics. Accumulating evidence suggests that Cre activity can, in addition to excising targeted loxP sites, induce cytotoxic effects, including abnormal cell cycle progression, genomic instability, and apoptosis, which can accelerate cancer progression. It is speculated that these defects are caused by Cre-induced DNA damage at off-target sites. Here we report the formation of tetraploid keratinocytes in the epidermis of keratin 5 and/or keratin 14 promoter-driven Cre (KRT5- and KRT14-Cre) expressing mouse skin. Biochemical analyses and flow cytometry demonstrated that Cre expression also induces DNA damage, genomic instability, and tetraploidy in HCT116 cells, and live-cell imaging revealed an extension of the G₂ cell cycle phase followed by defective or skipping of mitosis as cause for the tetraploidy. Since tetraploidy eventually leads to aneuploidy, a hallmark of cancer, our findings highlight the importance of distinguishing non-specific cytopathic effects from specific Cre/loxP-driven genetic manipulations when using Cre-mediated gene deletions.     

Reconstruction of mreB Expression in Staphylococcus aureus via a Collection of New Integrative Plasmids

Protein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial models Escherichia coli and Bacillus subtilis have been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacterium Staphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of the S. aureus chromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression of mreB in S. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that in S. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the use S. aureus as a model system in exploring diverse aspects of cellular microbiology.     

Gene cloning system for sulfonamide-mineralizing <Emphasis Type="Italic">Microbacterium</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> strain BR1

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli–Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.     

Gene 32 protein of bacteriophage T4 moderates the activities of the T4 gene 46/47-controlled nuclease and of the Escherichia coli RecBC nuclease in vivo.

Genes 46 and 47 of phage T4 control a nuclease that is required for genetic recombination and may act similarly to the Escherichia coli RecBC nuclease. In vivo, the nucleolytic activities of both of these nucleases must be moderated so that recombining DNA intermediates are not destroyed. We conclude from our present experiments that the phage T4 gene 32 protein, specifically its C-terminal domain, participates in such moderation. We have investigated DNA degradation in different gene 32 and gene 32/46 mutants under conditions that are completely restrictive for progeny production in all the mutants. Under these conditions, DNA of those gene 32 mutants in which the C-terminal domain of the protein is not synthesized or is modified is degraded to acid-soluble material. T4 gene 46 or E. coli recB mutations reduce such degradation; together they abolish it completely. By contrast, single gene 32 mutants which produce an unaltered C-terminal domain show little or no degradation of their DNA. Residual protection against nucleases is unrelated to residual primary DNA replication or to overproduction of the mutant peptides in the different gene 32 mutants.     

Technical approaches for mouse models of human disease

The mouse is the leading organism for disease research. A rich resource of genetic variation occurs naturally in inbred and special strains owing to spontaneous mutations. However, one can also obtain desired gene mutations by using the following processes: targeted mutations that eliminate function in the whole organism or in a specific tissue; forward genetic screens using chemicals or transposons; or the introduction of exogenous transgenes as DNAs, bacterial artificial chromosomes (BACs) or reporter constructs. The mouse is the only mammal that provides such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation, and is therefore a powerful application for modeling human disease. This poster review outlines the major genome manipulations available in the mouse that are used to understand human disease: natural variation, reverse genetics, forward genetics, transgenics and transposons. Each of these applications will be essential for understanding the diversity that is being discovered within the human population.     

Enhancement and inhibition by 2'-O-hydroxyethyl residues of gene targeting mediated by triple helix forming oligonucleotides

Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2'-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2'-O-hydroxyethyl (2'-HE) substitutions, in combination with other 2' modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2'-HE residues enhance the bioactivity of TFOs containing 2'-O-methyl (2'-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2'-O-aminoethyl (2'-AE) residues.     

Measuring the costs of reproduction

The measurement of costs of reproduction is of interest because such costs are generally assumed by life history theory. There is some controversy concerning how to measure costs: common methods include experimental manipulations of life history, such as preventing some individuals from reproducing, or estimates of genetic correlations. These two methods often yield similar results, suggesting that one can serve as a substitute for the other. There are now experiments which demonstrate that there are different mechanisms underlying the response to an experimental manipulation versus a genetic correlation, so the two methods are not equivalent in estimating costs.     

F9 embryonal carcinoma cells engineered for tamoxifen-dependent Cre-mediated site-directed mutagenesis and doxycycline-inducible gene expression

The study of gene functions in complex genetic environments such as mammalian cells would greatly benefit from systems allowing a tight control of gene expression. The tetracycline-inducible gene expression system and the site-specific Cre/loxP recombination system have gained increasing popularity for conditional expression and gene disruption. To facilitate the analysis of gene functions in a cell autonomous system, we have established an F9 murine embryonal carcinoma cell line, constitutively expressing both the doxycycline-controlled transactivator rtTA and the tamoxifen-dependent Cre recombinase Cre-ER(T). The expression of a reporter gene placed under the control of tetracycline operators was induced about 1000-fold by doxycycline, and tamoxifen-induced excision of a loxP-flanked DNA segment occurred in all cells. This genetically engineered cell line, which allows, upon simple ligand addition, sophisticated genetic manipulations, such as sequential inactivation of loxP-flanked genes, and tightly controlled reexpression of their cDNAs, should be a valuable tool for studying mammalian gene functions.     

Raman microspectroscopy of algal lipid bodies: β-carotene quantification

Advanced optical instruments can serve for analysis and manipulation of individual living cells and their internal structures. We have used Raman microspectroscopic analysis for assessment of β-carotene concentration in algal lipid bodies (LBs) in vivo. Some algae contain β-carotene in high amounts in their LBs, including strains which are considered useful in biotechnology for lipid and pigment production. We have devised a simple method to measure the concentration of β-carotene in a mixture of algal storage lipids from the ratio of their Raman vibrations. This finding may allow fast acquisition of β-carotene concentration valuable, e.g., for Raman microspectroscopy assisted cell sorting for selection of the overproducing strains. Furthermore, we demonstrate that β-carotene concentration can be proportional to LB volume and light intensity during the cultivation. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on the spectroscopic features of the individual living cells. The resultant apparatus could find its use in demanding biotechnological applications such as selection of rare natural mutants or artificially modified cells resulting from genetic manipulations.     

Designing humans: A human rights approach

Advances in genomic technologies such as CRISPR‐Cas9, mitochondrial replacement techniques, and in vitro gametogenesis may soon give us more precise and efficient tools to have children with certain traits such as beauty, intelligence, and athleticism. In this paper, I propose a new approach to the ethics of reproductive genetic engineering, a human rights approach. This approach relies on two claims that have certain, independent plausibility: (a) human beings have equal moral status, and (b) human beings have human rights to the fundamental conditions for pursuing a good life. I first argue that the human rights approach gives us a lower bound of when reproductive genetic engineering would be permissible. I then compare this approach with other approaches such as the libertarian, perfectionist, and life worth living approaches. Against these approaches, I argue that the human rights approach offers a novel, and more plausible, way of assessing the ethics of reproductive genetic engineering.     

Epidermal stem cells and ex vivo cutaneous gene therapy: application to xeroderma pigmentosum

Ex vivo cutaneous gene therapy is an alternative treatment for recessively inherited diseases with cutaneous traits. It relies on the transfer in cultured epidermal keratinocytes of the wild-type allele of the gene whose mutation is responsible for the disease. As for severely burnt patients, epithelial sheets developed from genetically corrected cells may then be grafted back to the patients. Long term correction and graft take depend on the genetic correction of stem cells. Success of such an approach has recently been reported in the case of one patient suffering from a severe case of junctional epidermolysis bullosae. Here we report a method for safely selecting keratinocytes populations after genetic manipulation. The method is non invasive and non immunogenic and allows high enrichment of genetically manipulated stem keratinocytes. This could perhaps contribute to ex vivo gene therapy approaches of cancer prone genodermatoses such as xeroderma pigmentosum.     

Therapeutic applications of CRISPR/Cas9 system in gene therapy

Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. The clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) system, derived from the acquired immune system in bacteria and archaea, has provided a new tool for accurate manipulation of genomic sequence to attain a therapeutic result. The advantage of CRISPR which made it an easy and flexible tool for diverse genome editing purposes is that a single protein (Cas9) complex with 2 short RNA sequences, function as a site-specific endonuclease. Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy. In this article, we review the fundamental mechanisms of CRISPR-Cas9 function and summarize preclinical CRISPR-mediated gene therapy reports on a wide variety of disorders.     

植物种子中脂肪酸代谢途径的遗传调控与基因工程

本文介绍了植物种子中脂肪酸代谢的基本途径,并从工业用和食用两方面对近年来植物脂肪酸代谢的遗传调控与基因工程研究的进展进行了较为详细的总结。植物种子中的脂肪酸成分可通过人为遗传调控发生改变。阐明利用基因工程技术调控植物种子脂肪酸代谢途径是一个新兴的、具有巨大潜力的研究领域,并将在人们生活的各方面发挥巨大作用。