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A Novel Gene Transmission Pattern of Exogenous DNA in Offspring Obtained After Testis-Mediated Gene Transfer (TMGT)
Masahiro Sato and Shingo Nakamura

We have attempted to establish a new method, so-called “testis-mediated gene transfer, TMGT”, based upon gene transfer via direct introduction of exogenous plasmid DNA into a testis as an alternative to microinjectionmediated transgenesis. We found that i) high transmission rate of exogenous DNA to offspring is achieved after TMGT, ii) the pattern of gene transmission from F0 to F2 generations is non-Mendelian, iii) the copy number of exogenous DNA is below 1 copy per diploid cell, and iv) gene expression does not occur or is very slight if it occurs. In this study, we examined the pattern of gene transmission of exogenous DNA in the offspring (at F0 to F2 generations) obtained after TMGT. A complex (70 ml) of circular pCAG/NCre plasmid (a Cre expression plasmid; 8 mg/testis) and FuGENE™6 (16 ml/testis; Boehringer Mannheim GmbH, Germany) was injected into the testes of transgenic mice carrying a loxP-flanked enhanced green fluorescent protein cDNA sequence (termed CETZ-17) 3 times 3 days apart. On 7 to 21 days after final injection, these injected males were mated to superovulated CETZ-17 females to obtain F0 pups. From these mice, F1 and F2 offspring were obtained. Genotyping of these mice was performed by PCR using several primers recognizing several parts of pCAG/NCre. We observed the following: i) at least two types of pCAG/NCre which included intact plasmid and deleted form (lacking pBluescript SK(-) vector backbone) of plasmid in F0 offspring, ii) during transition of F0 to F1, conversion of the intact form to the deleted one occurred frequently, and iii) the deletion appeared to occur outside the 1.3-kb NCre gene (probably at a portion containing CAG promoter and the 1st intron of the chicken b-actin gene, and a portion containing the 3’-noncoding region of the rabbit b-globin gene and SV40 poly(A) signals). These findings suggest i) the possible occurrence of autosomal proliferation of plasmid or its survival in mouse tissues during TMGT-mediated gene delivery, and ii) the presence of a regulated mechanism to elicit deletion of the exogenous plasmid pCAG/NCre probably at defined sites.

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