Sperm-mediated gene transfer: production of pigs transgenic for a human regulator of complement activation.
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Animals↗
Biomedical subjects
M Lavitrano
Publications and source records attributed to M Lavitrano.
A human neuroblastoma xenograft model for 125-I-metaiodobenzylguanidine biodistribution studies.
We developed an animal model to evaluate the 125-I-metaiodobenzylguanidine (125-I-mIBG) biodistribution in tumor bearing mice. Six weeks old nude-atimic mice were subcutaneously injected with 30 x 10(6) cells of the human neuroblastoma (NB) cell line SH-SY5Y. TE-671, a rhabdomyosarcoma cell line, was used as a control tumor without a specific mIBG uptake mechanism. In order to prevent possible tumor rejection mediated by NK activity the anti asialo GM1 antiserum was administered intraperitoneally once a week for 4 weeks. The maximum anti asialo mediated effect was obtained by administering the first dose the same day as the cell implant. In this group of animals by 9 weeks 98% of mice had a measurable tumor. We have utilized this model to evaluate the biodistribution of 125-I-mIBG given as two different formulations: standard preparation with a specific activity of 84 mCi/mg and the no carrier added (n.c.a) formulation with a specific activity of approximately 8,000 mCi/mg. Our preliminary results indicate that the biodistribution of the two different formulations in the various organs are similar. Therefore it appears that n.c.a. mIBG should not cause an increased toxicity in possible normal target organs such as heart or adrenals. Additional experiments will be performed in this model to ascertain if there is a potential advantage of the clinical use of n.c.a. mIBG over the standard preparation.
3-Iodobenzylguanidine↗
The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake.
Mature sperm cells have the spontaneous capability of taking up exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatoza. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind exogenous DNA. Thus, the interaction of exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.
Animals↗
Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells.
Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15-22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA.
Animals↗
The interaction between exogenous DNA and sperm cells.
Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.
Animals↗
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.
Animals↗
Human seminal ribonuclease. A tool to check the role of basic charges and glycosylation of a ribonuclease in the action of the enzyme on double-stranded RNA.
Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E. and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356-363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Results indicate (1) that glycosylation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) X poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.
Glycoproteins↗
Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer.
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Animals↗
Xenotransplantation: state of the art.
Organ transplantation is unfortunately limited by the number of cadaveric human donor organs that become available. Xenotransplantation - the transplantation of organs and tissues between animal species - would supply an unlimited number of organs and offer many other advantages. The leading candidate as a source of organs for xenotransplantation is the pig, as it is anatomically and physiologically similar to man. However, organ transplantation between distantly related species results in hyperacute rejection (HAR) of the transplant, which consists of a violent immune response involving the complement system, unleashed when organs are transplanted between distant relatives, such as pigs and humans. HAR destroys the blood vessels in the transplanted organs and kills them within minutes. Complement attack is due to the antibody-mediated activation of the complement cascade (human anti-pig antibodies have been identified as being directed against Gal-a1-3galactose epitopes on pig vascular endothelium), and this phenomenon is physiologically subjected to negative regulation by a set of species-specific proteins, known as regulators of complement activation (RCA), such as decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. If human RCA are expressed by mammalian cells in vitro, the cells are protected against the lysis by human complement. Therefore the incorporation of human RCA into a xenogeneic organ by transgenesis should protect the transplanted organ or tissue from in vivo lysis by human complement. Major efforts are being made to overcome this hyperacute rejection. Methods being investigated include: i. depletion or inhibition of recipient antibodies or complement; ii. development of immunological tolerance to pig organs in the recipient; and iii. development of transgenic pigs that do not express the a-Gal epitope and/or express a human complement inhibiting protein (i.e., DAF). A small number of research teams, including our group, have embarked on programs to produce transgenic pigs for one of the human RCA, in the attempt to produce animals whose organs may be suitable for transplantation into humans.
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Base composition of dromedary thymus DNA.
The base composition of dromedary thymus DNA was determined by reversed-phase HPLC determination of the four major deoxyribonucleosides. No significant differences were found between dromedary and calf thymus DNA. The elution system used (different from that suggested in the literature) was ammonium phosphate buffer/acetonitrile.
Animals↗