Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We have constructed a regulatable bidirectional adenovector expressing the humanized green fluorescent protein (GFP) and oct4, sox2, klf4, and c-myc genes (known as the Yamanaka genes or OSKM). The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem), which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the gFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly generated vector was expanded by six iterated coinfections of the above cells which were lysed at the end of the process and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of the initial preparation was 1.2 × 10¹² physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the four Yamanaka genes. Additionally, 3 days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced the GFP expression. This adenovector constitutes a promising tool for implementing nonintegrative partial cell reprogramming.

通过部分细胞重编程的生物复兴是新兴的研究途径。在这种情况下，可调节的多能性基因表达系统是目前使用最广泛的。我们构建了可调节的双向腺载体，表达人源化绿色荧光蛋白（GFP）和o ct4，sox2，klf4和c-myc基因（称为Yamanaka基因或OSKM）。OSKM基因排列为双顺反子串联（hSTEMCCA串联），它受Tet-Off双向启动子的控制，该启动子还控制gFP的表达基因。另外，组成型盒表达调节蛋白tTA。载体DNA被转染到HEK293 Cre细胞中，该细胞还被辅助腺载体H14感染，由于HEK293 Cre细胞产生的Cre重组酶而无法包装其DNA。新产生的载体通过上述细胞的六次重复合并感染而扩增，这些细胞在过程结束时被裂解，腺载体通过在CsCl梯度中超速离心进行纯化。初始制备的效价为1.2×10 ¹²物理病毒颗粒/毫升。如预期的那样，载体转导的大鼠成纤维细胞培养物中的GFP荧光随着培养基中存在的强力霉素（DOX）剂量而下降。转导细胞的免疫细胞化学分析证实了四个Yamanaka基因的表达。另外，在大鼠下丘脑中注射载体后3天，在该区域观察到显着水平的荧光。在饮用水中添加2 mg / ml DOX会降低GFP表达。该腺载体构成用于实施非整合性部分细胞重编程的有前途的工具。