A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis.gene therapy ͉ site-specific ͉ transposase ͉ cancer ͉ mutagenesis
Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6 mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6 mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6 mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.
The efficiency of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is often limited by poor embryo development. Because our previous work indicated that impairment of embryo development is frequently related to chromosomal abnormalities, we hypothesized that foreign DNA and/or conditions used to enhance integration of the DNA might induce chromosome damage. Therefore, we examined the chromosomes of mouse embryos produced by transgenesis with the EGFP gene. Spermatozoa were processed with three methods that cause membrane disruption: freeze-thawing, Triton X-100, or Triton X-100 followed by a sucrose wash. Membrane-disrupted spermatozoa were mixed with EGFP plasmids and injected into metaphase II oocytes. Three endpoints were evaluated: paternal chromosomes of the zygote, embryo capacity to develop in vitro, and expression of the transgene at the morula/blastocyst stage. In all pretreatments, we observed a significant decrease (approximately 2-fold) in the frequency of normal karyoplates when spermatozoa were incubated with exogenous DNA as compared with the treatment when no DNA was added. As predicted, embryo development was correlated with the integrity of the paternal chromosomes of the zygote. Searching for the possible mechanism of chromosome degradation, we used the ion chelators EGTA and EDTA and found that they neutralize the harmful effect of the transgene and stabilize the paternal chromosomes. In the presence of chelating agents, however, the number of embryos expressing EGFP produced with ICSI-mediated transgenesis decreased significantly. The results suggest that treatment of spermatozoa with exogenous DNA leads to paternal chromosome degradation in the zygote. Furthermore, the mechanisms of disruption of paternal chromosomes and the integration of foreign DNA may be closely related.
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