Pham Linh, Nakamura Takafumi, Gabriela Rosales A, Carlson Stephanie K, Bailey Kent R, Peng Kah-Whye, Russell Stephen J
Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
J Gene Med. 2009 Mar;11(3):197-206. doi: 10.1002/jgm.1289.
Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome.
Three expression cassettes, each comprising a cytomegalovirus promoter driving one of three marker peptides [serum carcinoembryonic antigen (sCEA), beta subunit of human chorionic gonadotropin (betahCG) or human sodium iodide symporter (hNIS)], were inserted into E1, E3 or E4 cloning sites in a recombinant adenoviral vector backbone. High titer stocks of bicistronic adenoviral vectors coding for combinations of marker peptides were prepared. A panel of human cells of various lineages was infected with the vectors and expression ratios of the transgene-encoded proteins were analysed. Serum levels of the soluble proteins and hepatic uptake of radioactive iodine were also compared in vivo in nude rats after intravenous vector infusion.
High concordance of expression between the inserted transgenes was observed in all of the bicistronic vectors irrespective of whether the expression cassettes were placed in the E1, E3 or E4 regions. Concordance was maintained across multiple cell lineages. In vivo, in athymic rats, blood and urine levels of betahCG were highly concordant with serum levels of sCEA at all timepoints after intravenous infusion of the bicistronic vectors encoding both of these soluble markers. Hepatic radioiodine uptake was concordant with serum CEA concentration in mice infused with a bicistronic vector expressing CEA and NIS.
The expression level of a given transgene in an adenoviral vector genome can be accurately and quantitatively inferred from the expression of a marker protein encoded by a second transgene inserted elsewhere in the vector genome.
表达盒可插入重组腺病毒基因组的多个位置，但这种选择对转基因表达水平的影响尚未确定。了解腺病毒基因组中不同位点插入的转基因的相对表达水平，对于依赖于插入病毒基因组其他位置的标记转基因的一致表达的转基因表达监测方法具有特别重要的意义。
将三个表达盒分别插入重组腺病毒载体骨架的E1、E3或E4克隆位点，每个表达盒都包含一个驱动三种标记肽之一[血清癌胚抗原（sCEA）、人绒毛膜促性腺激素β亚基（betahCG）或人钠碘同向转运体（hNIS）]的巨细胞病毒启动子。制备了编码标记肽组合的双顺反子腺病毒载体的高滴度病毒原液。用这些载体感染一组不同谱系的人类细胞，并分析转基因编码蛋白的表达比率。静脉注射载体后，还在裸鼠体内比较了可溶性蛋白的血清水平和放射性碘的肝脏摄取情况。
在所有双顺反子载体中，无论表达盒是置于E1、E3还是E4区域，插入的转基因之间均观察到高度一致的表达。在多个细胞谱系中均保持一致。在无胸腺大鼠体内，静脉注射编码这两种可溶性标记物的双顺反子载体后，在所有时间点，betahCG的血液和尿液水平与sCEA的血清水平高度一致。在注射表达CEA和NIS的双顺反子载体的小鼠中，肝脏放射性碘摄取与血清CEA浓度一致。
从载体基因组其他位置插入的第二个转基因编码的标记蛋白的表达，可以准确且定量地推断腺病毒载体基因组中给定转基因的表达水平。
