Control over the number of copies inserted at any one locus can be achieved by using retroviral vectors. Lin et al. (31) reported the generation of transgenic zebrafish using a pseudotyped retroviral vector. This vector, developed by Burns et al. (26), can be concentrated to very high titers and can infect cultured cells derived from zebrafish embryos. The virus contains an Moloney murine leukemia virus (MoMLV)-based genome surrounded by an envelope containing the glycoprotein (G-protein) of the vesicular stomatis virus (VSV), completely replacing the retroviral env glycoprotein. The presence of the VSV G-protein confers the same broad host range, which is characteristic of VSV, on the pseudotyped vector. On entry into a permissive cell, the vector integrates retroviral sequences into the host genome.
The pseudotyped virus, LZRNL(G), was injected into the blastoderm cells at the 2000-4000-cell stage. About 50-100 infectious units were injected/ embryo. Embryos were raised to sexual maturity, and DNA from pools of their 24-h-old F1 progeny was tested for the presence of LZRNL sequences by PCR. Eight of 50 founder fish showed germline transmission. Two transgenic F1 fish transmitted the viral sequences at 44% (11 out of 25) and 47% (8 out of 17) to their F2 progeny. Southern analysis showed that all founder fish had single copies at the point of insertion and that some founder fish had more than one retroviral insertion, which segregated independently in the F1. The viral construct had two reporter genes: lacZ fused to Moloney LTR, and neo driven by RSV-LTR. The former is not active in zebrafish cells. The neo gene has similarly not been shown to be expressed in these transgenic lines.
Gaiano et al. (33) constructed modified retroviral vectors that give high titers. When injected into 2000- to 4000-cell blastula, they give very high transgenic frequencies. In three experiments, 110 of 133 (83%) of the embryos injected were found to transmit proviral insertions through their germ line into the F1. In one of these three experiments, all 50 founder fish transmitted the proviral insertions to their F1 progeny. The difference being that the embryos were incubated at 28°C rather than 26°C. In two experiments, on average each positive founder transmitted transgene insertions to 29% of its F1 progeny. Each positive founder on average transmitted 11 proviral insertions to its F1 progeny. A second experiment with a different retroviral stock with a quarter of the titer of the first gave lower rates of transmission (13% compared with 29%). These significantly higher rates of generation of transgenic zebrafish reported by Gaiano et al. (33) are probably as a result of the approx 100-fold higher titers of retroviral vectors used.
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