Tran N D, Porada C D, Zhao Y, Almeida-Porada G, Anderson W F, Zanjani E D
Department of Veterans Affairs Medical Center, University of Nevada, Reno 89520, USA.
Exp Hematol. 2000 Jan;28(1):17-30. doi: 10.1016/s0301-472x(99)00133-2.
We have previously reported that directly injecting low-titer retroviral vector supernatant into pre-immune sheep fetuses resulted in the transfer and long-term expression of the bacterial NeoR gene within the hematopoietic system of these animals for over 5 years. In the present studies, we investigated whether using a higher titer vector would enable more efficient transduction and expression of the transgenes within the hematopoetic cells in sheep injected in utero.
Sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatant encoding the bacterial NeoR and LacZ genes (titer: 1x10(7) cfu/mL).
Over the 2-year time course of these studies, the presence and expression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained > or =6% transduced peripheral blood lymphocytes. Vector distribution was widespread without any detectable pathology. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered.
These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving long-term expression of the transgenes within the recipient's hematopoietic cells. Furthermore, expression of the NeoR gene from these studies was higher than that reported in our previous study in which a lower titer vector was used.
我们之前报道过，将低滴度逆转录病毒载体上清液直接注射到未免疫的绵羊胎儿体内，可使细菌新霉素抗性基因（NeoR）在这些动物的造血系统中转移并长期表达超过5年。在本研究中，我们调查了使用更高滴度的载体是否能使子宫内注射的绵羊造血细胞更有效地转导和表达转基因。
16只未免疫的绵羊胎儿经腹腔注射了编码细菌NeoR和LacZ基因的G1nBgSvNa8.1无辅助逆转录病毒载体上清液（滴度：1×10⁷ cfu/mL）。
在这些研究的2年时间里，通过多种免疫和生化方法对14只评估动物中的12只进行检测，证实了NeoR和LacZ基因的存在和表达。通过流式细胞术分析检测的12只绵羊中，有7只含有≥6%的转导外周血淋巴细胞。载体分布广泛，未发现任何可检测到的病理学变化。重要的是，PCR分析和繁殖实验表明种系未改变。
这些研究证实，直接注射工程化逆转录病毒是将外源基因安全导入发育中胎儿体内并从而在受体造血细胞中实现转基因长期表达的可行方法。此外，本研究中NeoR基因的表达高于我们之前使用较低滴度载体的研究报道。
