1 kb nucleotides (HA117 Seliciclib gene) was obtained and sequenced, which indicated that the recombined plasimid pAdTrack/HA117 was constructed successfully. The pAdTrack-HA117 was homologous recombined with BJ-Adeasy in E. coli. Then, the recombined Adeasy-HA117 plasmid was identified by Pac1 cutting. One 30 kb strap and one 4.5 kb strap could be seen by agarose gel electrophoresis, which proved that the homologous recombination was successful (Figure 1). Then, pAdeasy-HA117 was transfected into 293 cells. After two weeks, the transfected 293 cells became to be float from adherence observed by the GFP fluorescence intensity (Figure

2). At this time, the completed recombined adenovirus Ad5-HA117 was harvested. Figure 1 Gel screening of Adeasy-HA117 after digested by Pac I. After digeted with Pac I, Adeasy-HA117 produced 4.5 kb DNA strap, which proved that the homologous recombination was successful. M: DNA Marker; 1,2: Adeasy-HA117 Figure 2 The generation of recombinated adenovirus pAdeasy-HA117. Expression of fluorescence and most suitable adenovirus amount of infetected K562 cells The K562 cells had green fluorescent expression at 24 hours after infected

(Figure 3). It was found that the infection rate of adenovirus to K562 cells increased with the adenovirus amout increased. Both cells’ survival rate (exceeded 80%) and infection rate (reached 39.72%) were fairly well when MOI was 100. And the weak and dead cells increased selleck products obviously when MOI exceeded 100. So MOI 100 was chosen as the most suitable amount for

the further investigation (Table 1 and Figure 4). Figure 3 Fluorescent expression of K562 cells after transfected 24 hours. A:K562 cells; B: K562/Ad-HA117 cells expressed green fluorescence. Figure 4 The infection rates of K562 cells during different MOI detected by FCM. The infection rates were about 39.72%~64.3%. Immune system A: MOI = 100; B: MOI = 1000. Table 1 The rates of infection and survival of cell during different MOI   MOI   1 10 50 100 500 1000 Infection rates 0.47 ± 0.04 5.83 ± 0.07 10.65 ± 0.11 16.19 ± 0.31 20.27 ± 0.52 30.42 ± 2.31 Survivil rates 90.33 ± 1.21 85.27 ± 1.37 82.11 ± 1.63 81 ± 1.42 62.23 ± 2.15 40.25 ± 2.13 RT-PCR results for HA117 gene expression in k562 cells Both uninfected K562 cells and K562/Ad-null cells had no HA117 gene expression, and HA117 expressed only in the K562/Ad-HA117 cells, which indicated that K562 cells could express exogenous HA117 gene when infected by Ad-HA117 (figure 5). Figure 5 The expression of HA117 gene mRNA in K562 cells. M: DNA marker; 1:K562 cells; 2: K562/Ad-null cells had no HA117 gene expression; 3:K562/Ad-HA117 cells had HA117 gene expression. The DNA strap having 397 bp was β-actin. The MTT assays results for K562 cells’ drug sensitivity The survival rates of K562/HA117 cells increased than that of K562 cells and K562/Ad-null cells. The RFs of K562/Ad-HA117 cells to VCR, ADM, Vp-16, DNR, MMC and CTX were 4.