Marialuisa Lavitrano

Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice

Mature mouse sperm cells incubated in an isotonic buffer with cloned DNA capture DNA molecules over a 15 min period. Spermatozoa incubated with pSV2CAT plasmid in either circular or linear form were used to fertilize mouse eggs in vitro. Sequences complementary to pSV2CAT were identified in approximately 30% of 250 progeny by Southern blotting. A genomic library was constructed from the DNA of a positive mouse. Three positive clones were identified and two adjacent HincII restriction fragments of 240 and 370 bp showed identical sequences to the corresponding fragments of the pSV2CAT plasmid. F1 progeny showed paternal and maternal transmission of the transgenes from founders. CAT gene expression was detected on tissues of adult F1 individuals, preferentially on tails and muscle. We conclude that transgenic mice can be obtained using sperm cells as foreign DNA vectors.

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.

'Advanced' generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo.

Efficient gene transduction in cardiomyocytes is a task that can be accomplished only by viral vectors. Up to now, the most commonly used vectors for this purpose have been adenoviral-derived ones. Recently, it has been demonstrated that lentiviral vectors can transduce growth-arrested cells, such as hematopoietic stem cells. Moreover, a modified form of lentiviral vector (the ‘advanced’ generation), containing an mRNA-stabilizer sequence and a nuclear import sequence, has been shown to significantly improve gene transduction in growth-arrested cells as compared to the third-generation vector. Therefore, we tested whether the ‘advanced’ generation lentivirus is capable of infecting and transducing cardiomyocytes both in vitro and in vivo, comparing efficacy in vitro against the third-generation of the same vector. Here we report that ‘advanced’ generation lentiviral vectors infected most (>80%) cardiomyocytes in culture, as demonstrated by immunofluorescence and FACS analyses: in contrast the percentage of cardiomyocytes infected by third-generation lentivirus was three- to four-fold lower. Moreover, ‘advanced’ generation lentivirus was also capable of infecting and inducing stable gene expression in adult myocardium in vivo. Thus, ‘advanced’ generation lentiviral vectors can be used for both in vitro and in vivo gene expression studies in the cardiomyocyte.

Sperm-mediated gene transfer

Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.

Sperm-mediated gene transfer in mice

Sperm‐mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm‐mediated DNA transfer to mouse eggs in well‐established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm‐mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm‐mediated transfer of foreign DNA. Mol. Reprod. Dev. 50:406–409, 1998.

Genetically modified pigs produced with a nonviral episomal vector

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

Sperm‐mediated DNA transfer in bovine and swine species

Abstract Ejaculated sperm cells from both bovine and swine animals depleted of the seminal fluid were used as vectors for transferring different plasmid DNAs into eggs during the fertilization process. PCR screening of about 2000 blastocysts showed that transformed embryos were obtained in both animal species with varying efficiencies, and that the rate of transformation depended on the plasmid used. The most efficient rate was obtained in both species with the pSV2CAT construct, yielding 22% positive blastocysts in bovine and 5.7% in swine respectively. Artificial insemination of ten sows with boar sperm cells that had been preincubated with pSV2CAT DNA produced 82 offspring. Southern blot analysis of the DNAs extracted from the animal tails showed that five animals were transgenic and contained sequences complementary to pSV2CAT DNA that appeared to be rearranged compared to the original plasmid. These results indicate that sperm‐mediated DNA transfer protocols can be successfully adapted for the genera...

Carbon monoxide improves cardiac energetics and safeguards the heart during reperfusion after cardiopulmonary bypass in pigs

Ischemia‐reperfusion injury, a clinical problem during cardiac surgery, involves worsened adenosine trisphosphate (ATP) generation and damage to the heart. We studied carbon monoxide (CO) pretreatment, proven valuable in rodents but not previously tested in large animals, for its effects on pig hearts subjected to cardiopulmonary bypass with cardioplegic arrest. Hearts of CO‐treated pigs showed significantly higher ATP and phosphocreatine levels, less interstitial edema, and apoptosis of cardiomyocytes and required fewer defibrillations after bypass. We conclude that treatment with CO improves the energy status, prevents edema formation and apoptosis, and facilitates recovery in a clinically relevant model of cardiopulmonary bypass surgery.

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi‐organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well‐characterized model of LPS‐induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS‐induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL‐1β in response to LPS, while augmenting the anti‐inflammatory cytokine IL‐10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS‐induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Adenoviral RB2/p130 Gene Transfer Inhibits Smooth Muscle Cell Proliferation and Prevents Restenosis After Angioplasty

Smooth muscle cell (SMC) proliferation that results in neointima formation is implicated in the pathogenesis of atherosclerotic plaques and accounts for the high rates of restenosis that occur after percutaneous transluminal coronary angioplasty, a widespread treatment for coronary artery disease. Endothelial lesions trigger intense proliferative signals to the SMCs of the subintima, stimulating their reentry into the cell cycle from a resting G(0) state, resulting in neointima formation and vascular occlusion. Cellular proliferation is negatively controlled by growth-regulatory or tumor-suppressor genes, or both, such as the retinoblastoma gene family members (RB/p105, p107, RB2/p130). In the present study, we show that RB2/p130 inhibited SMC proliferation in vitro and in vivo. We used the rat carotid artery model of restenosis to demonstrate that adenovirus-mediated localized arterial transduction of RB2/p130 at the time of angioplasty significantly reduced neointimal hyperplasia and prevented restenosis. Furthermore, the ability of pRb2/p130 to block proliferation correlated with its ability to bind and sequester the E2F family of transcription factors, which are important mediators of cell cycle progression. These results imply that RB2/p130 could be an important target for vascular gene therapy.