Liposome‐mediated gene transfer via sperm cells. High transfer efficiency and persistence of transgenes by use of liposomes and sperm cells and a murine amplification element

Abstract

Summary Plasmid DNA was encapsulated in liposomes to protect from degradation and to support the incorporation into sperm cells. Transgenic spermatozoa carry the foreign DNA to the target cell when fertilizing the egg. Several plasmid constructs were transferred into rabbits, cattle and chicken. The plasmid apparently can persist when provided with a murine amplification promoting sequence. The transgeneity of sperm cells as well as fetuses or offspring was demonstrated by dot blot, Southern blot analysis and PCR. Expression of the transgene was shown as enzymatic activities of the reporter genes SEAP or β‐galactosidase and by gelelectrophoretic characterization of the expressed gene products. The transfer efficiency and the persistence of the gene constructs is significantly increased due to the use of sperm cells as gene carriers mediated by liposomes and the amplification element. Zusammenfassung Gentransfer mittels Liposomen und Spermien Plasmid‐DNA wurde in Liposomen eingeschlossen, um sie vor enzymatischem Abbau zu schützen und ihre Inkorporation in die Spermienzelle zu ermöglichen. Bei der Befruchtung bringt das Spermium die Fremd‐DNA in die Eizelle. Es wurden verschiedene Genkonstrukte in Kaninchen, Rind und Huhn übertragen. Das Plasmid kann offensichtlich persistieren, wenn es eine murine amplifikationsfördernde Sequenz enthält. Die Transgenität von Spermien, Feten und Nachkommen wurde mit Dot Blot, Southern Blot und PCR nachgewiesen, die Expression der Reportergene SEAP und β‐Galaktosidase gelelektrophoretisch bzw. enzymatisch charakterisiert. Der Einsatz von Spermien und Liposomen zur Genübertragung sowie die Verwendung einer ‘amplification promoting sequence' erhöhen die Transfereffizienz und die Persistenz der Genkonstrukte beträchtlich.

Production of Transgenic Rats and Mice by the Testis-Mediated Gene Transfer

“…For example, Bachillar et al [32] reported that liposome-encapsulated foreign DNA can be incorporated into mouse sperm head after culture with spermatozoa isolated from the epididymis, though transgenic animals could not be generated by the sperm. However, the property of transporting exogenous DNA into the oocyte via sperm has been recently described in several species [12][13][14][15][16][17][18]. Together with the present data, these results indicate that sperm cells can be used as a vector transferring the gene under certain experimental condition.…”

“…By this conventional microinjection method, we have already produced 2 lines of transgenic rats expressing human growth hormone (hGH) gene under the control of mouse whey acidic protein (WAP) promoter [4][5][6]. However, it requires spe- [9][10][11], successful sperm-mediated gene transfer has been now reported in a variety of animal species including mollusk [12], insect [13], fish [14], bird [15] and mammals [16][17][18]. It would be a more useful alternative, especially on laboratory animals such as rats and mice, if an injection of DNA into the testis could generate transgenic animals via sperm ejaculated (testis-mediated gene transfer).…”

Protamine-Derived Synthetic Peptide Enhances the Efficiency of Sperm-Mediated Gene Transfer Using Liposome-Peptide-DNA Complex.

“…One of the more convenient alternatives is sperm-mediated gene transfer (SMGT), which uses spermatozoa as vectors for DNA delivery [5]. A large number of transgenic animals including mice, pigs and cattle have been produced by SMGT using sperm associated with naked DNA or liposome-DNA complex [6][7][8][9][10][11][12]. However, the efficiency and reliability of SMGT are still controversial [13,14], though the capacity of spermatozoa capturing foreign DNA has been proven [15,16].…”

Possible Mechanisms for the Testis-Mediated Gene Transfer as a New Method for Producing Transgenic Animals.

“…Since the pioneering study by Brackett et al [1] showing that rabbit spermatozoa were able to incorporate SV40 DNA into their heads during incubation, the property of association of spermatozoa with heterologous DNA has been extensively studied [2][3][4][5]. These studies have facilitated the idea of using sperm as a vector to transfer exoge-actually carry the DNA into eggs, and the gene thus transferred is transcriptionally active [11][12][13][14][15][16][17]. In addition, as described in the associated paper [18], we have shown that DNAs injected into the testis of either rats or mice can be transferred into eggs via sperm at fertilization (testis-mediated gene transfer).…”