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PubMed1996

[Waardenburg's syndrome].

Waardenburg syndrome is an autosomal dominant disorder characterised by sensorineural hearing loss, pigmentary abnormalities of iris, hair and skin and dystopia canthorum. The expression of clinical findings of Waardenburg syndrome is extremely variable. The potential sources of this variability include allelic variation, genetic heterogeneity, epistatic modifying genes and stochastic effects. This type of multifactorial inheritance based on oligogenic epistasis is very different from the model based on polygenic inheritance. Indeed in the first model, knowledge of the genotype at relevant modifier loci may allow prediction and possibly treatment of clinically relevant aspects of the phenotype. Therefore a more complete understanding of gene expression in Waardenburg could provide insights that are relevant to many other multifactorial diseases.

Animals↗
PubMed2006

Evaluation of potential modifiers of the palatal phenotype in the 22q11.2 deletion syndrome.

OBJECTIVE: To evaluate potential modifiers of the palatal phenotype in individuals with the 22q11.2 deletion syndrome. DESIGN: Data from 356 subjects enrolled in a study of the 22q11.2 deletion syndrome were used to evaluate potential modifiers of the palatal phenotype. Specifically, subjects with and without velopharyngeal inadequacy and/or structural malformations of the palate were compared with respect to gender, race, and genotype for variants of seven genes that may influence palatal development. METHODS: The chi-square test or Fisher exact test was used to evaluate the association between palatal phenotype and each potential modifier. Odds ratios and their associated 95% confidence intervals were used to measure the magnitude of the association between palatal phenotype, subject gender and race, and each of the bi-allelic variants. RESULTS: The palatal phenotype observed in individuals with the 22q11.2 deletion syndrome was significantly associated with both gender and race. In addition, there was tentative evidence that the palatal phenotype may be influenced by variation within the gene that encodes methionine synthase. CONCLUSIONS: Variation in the palatal phenotype observed between individuals with the 22q11.2 deletion syndrome may be related to personal characteristics such as gender and race as well as variation within genes that reside outside of the 22q11.2 region.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2003

Prevention of hepatocellular carcinoma in mice by IL-2 and B7-1 genes co-transfected liver cancer cell vaccines.

AIM: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice. METHODS: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards. The immunoprotection was investigated and the reactive T cell line was assayed. RESULTS: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes co-transfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7 vaccine) on the onset of tumor formation was the strongest. When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, chi2=7.70-11.69, P<0.05) and the implanted tumor was the smallest (z =3.20-44.10, P<0.05). The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively. The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P<0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5+/-2.5 %, 65.0+/-2.9 %, 83.1+/-1.5 % respectively, compared with other groups, P<0.05). CONCLUSION: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastasis of HCC.

Animals↗
PubMed2005

Efficient gene transfer to human epidermal keratinocytes on fibronectin: in vitro evidence for transduction of epidermal stem cells.

The epidermis is an attractive target for gene therapy because it is easily accessible and has great potential as an ectopic site for protein delivery in vivo. Genetically modified keratinocytes can be expanded in culture and used to generate three-dimensional skin equivalents, which can deliver therapeutic proteins either locally or systemically for the treatment of wounds or systemic diseases. Here we present an optimum protocol that yields consistently high retroviral gene transfer on a substrate of recombinant fibronectin (rFN). Gene transfer on rFN depends strongly on virus concentration and the density of target cells. Interestingly, the kinetics of gene transfer varies depending upon the origin--mouse or human--of virus-producer cells. Most notably, long-term growth and clonogenic assays show that transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. In contrast, the traditional protocol transduces mostly differentiated keratinocytes. We also show that skin equivalents prepared from genetically modified keratinocytes display high levels of transgene expression, mainly in the suprabasal layers. Our results are important for cutaneous gene therapy and for biological studies that require efficient and permanent genetic modification.

Animals↗
PubMed1994

Down-regulation of P4501A1 and P4501A2 mRNA expression in isolated hepatocytes by oxidative stress.

We have previously shown that the inflammatory mediator interleukin-1 suppressed transcription of CYP1A1 and CYP1A2 mRNAs (Barker, C.W., Fagan, J.B., and Pasco, D.S. (1992) J. Biol. Chem. 267, 8050-8055). Since many of the actions of inflammatory mediators are mimicked by oxidative stress, we treated isolated hepatocytes with 0.25-1.0 mM H2O2 to determine whether expression of these genes is also modulated by oxidative stress. Inducer-dependent accumulation of CYP1A1 and CYP1A2 mRNAs were maximally reduced approximately 50 and 70%, respectively, by 1.0 mM H2O2. Run-on transcription analysis suggested that the effect of H2O2 was mediated transcriptionally. The reduction in CYP1A mRNA levels was not due to a reduction in the levels of all mRNAs due to some general toxic effect since H2O2 did not reduce glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, beta-fibrinogen, or albumin mRNA levels, and did not increase lactate dehydrogenase released into the medium. Insulin-mimicked H2O2 action, reducing the expression of both mRNAs, and N-acetylcysteine, which increases intracellular glutathione levels, completely reversed the insulin effect on both mRNAs and the H2O2 effect on CYP1A1 mRNA, but only partially reversed the H2O2 effect on CYP1A2 mRNA. This study indicates that the CYP1A1 and CYP1A2 genes are responsive to oxidative stress and that the majority of this responsiveness can be modified by cellular redox potential.

Animals↗
PubMed1998

Crosstalk between alpha-1A and alpha-1B adrenoceptors in neonatal rat myocardium: implications in cardiac hypertrophy.

The myocardial effect of alpha-1A adrenoceptor (alpha-1 AR) agonists in neonatal rats are mediated by alpha-1A AR and not by alpha-1B AR, although both receptor subtypes are equally expressed; the functions of alpha-1B AR are not known. Here, we report that alpha-1 B ARs inhibit the activities of alpha-1A ARs in neonatal rat myocardium so that the inactivation of alpha-1 B ARs by chloroethylclonidine (CEC) potentiated the effects of nonselective alpha-1 AR agonist phenylephrine (PE) on myocardial protein synthesis and early gene (c-fos and c-jun) expression. CEC did not modify the hypertrophic effect of angiotensin II. The potentiation of the effects of PE by CEC was associated with a translocation of Ca(++)-dependent protein kinase C (PKC)alpha, which did not occur in the absence of CEC. Alpha-1A AR-selective agonist A61603 was approximately 1000-fold more potent than PE as a positive inotropic agent; it caused the translocation of PKC alpha, which was not affected by CEC. 5-Methylurapidil antagonized the effects of PE and A61603, suggesting that these were mediated via alpha-1A ARs. Alpha-1D AR antagonist BMY 7378 did not modify PE-induced translocation of PKC. CEC potentiated the effects of PE on Ca++ transients in Fura 2-AM-loaded dispersed cardiomyocytes, and this potentiation was prevented by nifedipine. In whole-cell patch-clamp recordings of cultured cardiomyocytes, CEC potentiated the effect of norepinephrine on Ca++ channel currents, which was blocked by 5-methylurapidil. We conclude that alpha-1A ARs are positively and alpha-1B ARs are negatively coupled to nifedipine-sensitive Ca++ channels, possibly via Gi protein, and this antagonistic relationship between alpha-A AR and alpha-1B AR in the neonatal heart might be required physiologically for normal alpha-1 AR-mediated responses and myocardial development.

Animals↗
PubMed2000

A recombinant vaccinia virus containing the papilloma E2 protein promotes tumor regression by stimulating macrophage antibody-dependent cytotoxicity.

Human papillomavirus infection is associated with cervical cancer. The E6 and E7 papillomavirus proteins are normally required for the maintenance of the malignant phenotype. Expression of these proteins in infected cells is negatively regulated by the binding of the papilloma E2 protein to the long terminal control region of the papilloma virus genome. The E2 protein can also promote cell arrest and apoptosis in HeLa cells. Therefore, it is clear that this protein has the potential of inhibiting the malignant phenotype. Because, anticancer vaccines based in vaccinia viruses have recently been shown to be an effective way to treat and to eradicate tumors, a recombinant vaccinia virus expressing the E2 gene of bovine papilloma virus (Modified Vaccinia Ankara, MVA E2) was created, to explore further the antitumor potential of the E2 protein. A series of rabbits, containing the VX2 transplantable papilloma carcinoma, were treated with MVA E2. An impressive tumor regression, up to a complete disappearance of tumor, was observed in most animals (80%). In contrast, very little or no regression was detected if the normal vaccinia virus was used. Lymphocytes isolated from MVA E2-treated rabbits did not show cytotoxic activity against tumor cells. However, in these animals a humoral immune response against tumor cells was observed. These antitumor antibodies were capable of activating macrophages to destroy tumor cells efficiently. These data indicate that injecting the MVA E2 recombinant vaccinia virus directly into the tumor results in a robust and long-lasting tumor regression. Data also suggest that antitumor antibodies are responsible, at least in part, for eliminating tumors by activating macrophage antibody-dependent cytotoxicity.

Amino Acid Sequence↗
PubMed2000

Safe and efficient gene transfer into porcine hepatocytes using Sendai virus-cationic liposomes for bioartificial liver support.

Establishment of a bioartificial liver support system using genetically modified hepatocytes is a potential approach to improve the treatment of severe liver failure. We describe the development of an efficient ex vivo method of gene transfer into a large number of porcine hepatocytes using hemagglutinating virus of Japan (HVJ)-liposome. The transfection efficiency of HVJ-liposome into isolated porcine hepatocytes attached to microcarrier beads was evaluated by beta-galactosidase (beta-gal) staining, fluorescence activated cell sorting analysis for beta-gal and luciferase assay, respectively. To examine the function and cellular damage of transduced hepatocytes, we used enzyme-linked immunosorbent assay for porcine albumin synthesis, lidocaine clearance test (P-450 activity), aspartate aminotransferase, and lactic dehydrogenase release assays. The optimal conditions for gene transfer into the beads-attached hepatocytes using HVJ-liposome included 4 microg of deoxyribonucleic acid with 200 microg of lipid/2 x 105 cells and exposure duration of 90 min. Under these conditions, beta-gal and luciferase genes were transduced to 2.5 x 108 isolated porcine hepatocytes following attachment to the beads. Positive beta-gal staining was observed in more than 30% of the beads-attached hepatocytes. The gene transfer activity of HVJ-liposome method determined by luciferase activities was about 100-fold of that of the lipofection method. Transfected porcine hepatocytes remained functional without any significant cell damage. Our results demonstrated that HVJ-liposome mediated gene transfer into microcarrier-attached porcine hepatocytes is an efficient and nontoxic method suitable for a bioartificial liver support sytem.

Animals↗
PubMed1997

Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice.

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase [1]; this generally requires 1-2 months of work for each construct. The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector. This approach leads to inactivation of the gene of interest [2]. Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing [3]. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. Our approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs [4-8], simplifying this technically demanding method.

Animals↗
PubMed2006

Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PURPOSE: Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. METHODS: Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. RESULTS: Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. CONCLUSIONS: Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Adhesives↗
PubMed1995

Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus.

OBJECTIVE: The aim was to examine the feasibility, efficiency and safety of adenovirus-mediated in vivo gene transfer into the canine myocardium by a percutaneous transluminal method using a needle-catheter. METHODS: Either a replication-defective adenovirus (Adex1SRLacZL) or a plasmid (pSRLacZ), both expressing E. coli lacZ coding beta-galactosidase (beta-gal), was directly injected into the left ventricle of dogs through a needle-catheter inserted via a femoral artery. Expression of lacZ was examined by histochemical staining and quantified by measuring beta-gal activity. RESULTS: Injections with Adex1SRLacZL induced lacZ expression as a result of 40 out of 41 injections; the expression level was 10 times higher than that obtained with pSRLacZ. Induced beta-gal activity was detected within 24 h, peaked at 7 days and retained for 2 weeks after gene transfer. A repetitive administration of the same adenovirus at 14 days after the first injection also evoked a reduced but significant level of expression despite neutralizing antibodies to adenovirus in serum. Although injection induced an inflammatory response that peaked at 3 days after injection and gradually subsided without a second peak, the temporal change and the extent of inflammation induced by adenovirus injection was not significantly different from those induced by injection with either saline or plasmid. Neither leakage of enzymes such as CPK or LDH nor alteration in the ECG was detected in the 30 days following gene transfer. CONCLUSIONS: Our findings demonstrate that a catheter-mediated direct injection with an adenovirus can induce gene expression in the ventricle more efficiently without additional myocardial damage and inflammation compared with injection with a plasmid. A repeat dose of the same adenovirus elicited gene expression at an attenuated but significant level. This method may potentially have clinical applications: in modifying myocardial phenotype and/or improving general circulation under certain circumstances.

Adenoviridae↗
PubMed2003

Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery.

Core shell silica particles with a hydrodynamic diameter of 28nm, an IEP of 7.1 and a zeta potential of +35mV at pH 4.0 were synthesised. The role of freeze-drying for the conservation of zwitterionic nanoparticles and the usefulness of different lyoprotective agents (LPA) for the minimisation of particle aggregation were studied. The activity of the nanoparticles was measured as DNA-binding capacity and transfection efficiency in Cos-1 cells before and after lyophilisation. It was found that massive aggregation occurred in the absence of LPA. Of the various LPAs screened in the present investigations, trehalose and glycerol were found to be well suited for conservation of cationically modified silica nanoparticles with simultaneous preservation of their DNA-binding and transfection activity in Cos-1 cells.

Animals↗
PubMed2004

Nerve growth factor: from animal models of cholinergic neuronal degeneration to gene therapy in Alzheimer's disease.

Over the last 20 years it has been recognized that neurotrophic factors profoundly influence the development of the nervous system and have the potential to modify disease processes in the adult nervous system. The ability of nervous system growth factors to prevent or reduce neuronal degeneration in animal models of neurodegenerative diseases has led to several clinical trials. One of the main obstacles to the success of these trials has been the method of growth factor delivery: sufficiently high doses of neurotrophic factors must be achieved in the target region of the brain to efficiently modify disease processes, but delivery must be restricted to specific brain regions to prevent adverse effects. Recent advances in molecular medicine have made gene therapy in the nervous system a potentially realistic approach for the delivery of therapeutic molecules such as growth factors. As an alternative to conventional drug delivery, several gene therapy trials for the treatment of central nervous system diseases have started or will start in the near future. This chapter reviews the development of neurotrophic factor gene therapy for neurodegenerative diseases focusing on the therapeutic potential of nerve growth factor in Alzheimer's disease, currently the subject of a phase I clinical trial.

Alzheimer Disease↗
PubMed1996

Stromal cells maintain the radioprotective capacity of CFU-S during retroviral infection.

Retroviral vectors provide an efficient means to introduce genes into hematopoietic stem cells. In order to develop retroviral infection protocols which preserve the radioprotective capacity of CFU-S, we designed a clonal hematopoietic reconstitution assay. In this assay, single CFU-S-derived derived colonies from bone marrow cells of 5-FU-treated mice were tested for their capacity to prevent radiation-induced mortality. Three parameters which may modify stem cell potential were tested in infection protocols using a retroviral vector containing the gene for neomycin resistance: (1) the partition of stem cells between the adherent and nonadherent fraction; (2) the replacement of the packaging cell line by a "competent' stromal cell line; and (3) the effects of G418 selection. All CFU-S having radioprotective capacity were found in the adherent fraction when the packaging cell line or the stromal cell line (MS-5) chosen for its capacity to maintain long-term bone marrow culture were used during the co-culture. The neo resistance gene was transduced into CFU-S with the same efficiency using co-culture with the packaging cell line or co-culture with the MS-5 cell line plus viral supernatant. However, in the presence of MS-5, a much higher proportion of CFU-S (70% versus 30%) had radioprotective properties, suggesting an important role for the stromal cells in the maintenance of hematopoietic reconstituting ability. Finally, G418 selection, even for a limited period (24 h), significantly decreased the radioprotective capacities of CFU-S (56% versus 18%). Subsequently, hematopoietic reconstitution by single CFU-S was quantified in recipient mice. The progeny of CFU-S were found at a significant level in the blood, spleen and bone marrow in 38% and 15% of mice, 1 and 3 months after transplantation, respectively. These results demonstrate that we have substantially improved the infection protocol. Under these conditions of infection, it is possible to conserve CFU-S properties and to transduce a gene into a stem cell with short-term hematopoietic reconstitution potential.

Animals↗
PubMed1999

Cellular delivery of hammerhead ribozymes conjugated to a transferrin receptor antibody.

Chemically-modified hammerhead ribozymes are sequence-specific RNA enzymes that can cleave target mRNA. These molecules have potential application as biological tools to understand gene expression and as therapeutic agents for the selective down-regulation of genes implicated in disease. However, as a result of their polyanionic character and relatively large molecular weights, ribozyme delivery to target cells is relatively inefficient. Using nuclease resistant 2'-O-methyl-modified ribozymes targeting the c-erbB1 oncogene, we have evaluated the potential use of human monoclonal transferrin-receptor antibody (TRA)-ribozyme conjugates for the improved delivery of ribozymes to A431 tumour cells. A 37-mer ribozyme derivatized with a free thiol-group at the 5'-end and bearing an internal [32P]-radiolabel was conjugated to either TRA or a non-specific IgG antibody using the heterobifunctional crosslinker, succinimidyl 4-(maleimido methyl)cyclohexane-1-carboxylate (SMCC). Up to six molecules of the ribozyme could be conjugated to one molecule of antibody. Cellular uptake studies in cultured human epidermoid A431 carcinoma cells showed that approximately a three-fold increase in cellular association could be obtained with the TRA-ribozyme conjugate compared to the free ribozyme. Cellular association of the conjugate was temperature-dependent and was inhibited by competition with excess free transferrin receptor antibody implying that conjugate uptake was consistent with the transferrin receptor-mediated endocytosis pathway. Treatment of cells with monensin further enhanced TRA-ribozyme conjugate cell association indicating that ribozyme delivery of conjugates may be further improved by strategies that modulate vesicular trafficking in cells.

Animals↗
PubMed2001

Diet-gene interactions in estrogen-induced mammary carcinogenesis in the ACI rat.

It is well accepted that hormonal, dietary and genetic factors each influence breast cancer risk. However, the underlying mechanisms and the extent to which these factors interact are largely unknown. We have demonstrated that the female ACI rat exhibits a unique genetically conferred propensity to develop mammary cancers when treated with physiological levels of 17beta-estradiol (E2). More recently, we have mapped to rat chromosome 5 a strong genetic modifier of susceptibility to E2-induced mammary cancers, termed estrogen-induced mammary cancer 1 (Emca1), and have identified potential Emca1 candidate genes. Because estrogens have been inextricably linked to the genesis of breast cancer in humans, the ACI rat model has the potential to reveal novel physiologically relevant insights into how the contributory actions of E2 are modified by specific dietary factors. In the present study, we have examined the ability of a 40% restriction of dietary energy consumption to inhibit E2-induced mammary carcinogenesis. The hypothesis tested was that energy restriction will inhibit mammary carcinogenesis even when circulating E2 remains elevated through administration of exogenous hormone. The data presented herein strongly suggest that energy restriction inhibits E2-induced mammary carcinogenesis in the ACI rat at least partly by retarding progression of atypical hyperplastic foci to carcinoma.

Animals↗
PubMed2001

Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection.

Chloroplast genetic engineering offers several advantages over nuclear transformation including high levels of gene expression and gene containment. However, a consequence of placing a transgene in the chloroplast genome is that the antibiotic resistance genes used as selectable markers are highly amplified. Engineering genetically modified (GM) crops without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes. Therefore, the betaine aldehyde dehydrogenase (BADH) gene from spinach was used in this study as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant. Chloroplast transformation efficiency was 25-fold higher in BA selection than with spectinomycin. In addition, rapid regeneration was obtained. Transgenic shoots appeared within 12 days in 80% of leaf disks (up to 23 shoots per disk) under BA selection compared to 45 days in 15% of disks (1 or 2 shoots per disk) under spectinomycin selection. Southern blots confirmed stable integration of foreign genes into all of the chloroplast genomes (approximately 10,000 copies per cell) resulting in homoplasmy. Transgenic tobacco plants showed 15- to 18-fold higher BADH activity at different developmental stages than untransformed controls. Transgenic plants were morphologically indistinguishable from untransformed plants and the introduced trait was inherited stably in the subsequent generation. This is the first report of genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. The use of naturally occurring genes in spinach for selection, in addition to gene containment, should ease public concerns regarding GM crops.

Alcohol Oxidoreductases↗
PubMed2004

Safety concerns related to hematopoietic stem cell gene transfer using retroviral vectors.

Endogenous retroviruses have developed efficient methods during their life cycle for stable integration into the host genome. Because of this ability, retroviral vectors were designed with the goal of gene transfer into hematopoietic stem cells (HSCs). The ability to genetically modify HSCs provides a vehicle for durable expression of potentially therapeutic transgenes in all lineages of mature blood cells for the lifetime of the patient. Combined with bone marrow transplant, retroviral gene transfer has many potential applications for a wide range of blood diseases. Advances in the development of oncoretroviral vectors based on murine leukemia viruses (MLV) and more recent development of human immunodeficiency virus (HIV)-based vectors have greatly increased the gene transfer efficiency. Optimization of methods for gene transfer using MLV-based vectors has substantially improved marking levels in mice, with lower levels in large animals and in human clinical trials. With advances in gene transfer technology has also come renewed concern about insertional mutagenesis and activation of oncogenes. Advanced techniques for integration site analysis combined with sequence comparison using mouse and human genome databases has now made it possible to begin to understand the spectrum of possible integration sites for both MLV- and HIV-based vectors. Furthermore, other studies have shown positive and negative dosage-dependent effects of transgene expression in mouse and human cells. Therefore, vector design and safety testing are at the forefront of the field of gene therapy and this review discusses recent developments.

Animals↗