In our previous studies, dozens of wild-type mice have received hydrodynamic injections, with or without integrase 
. Of these mice, as well as many more mice treated similarly in lab studies that have not been published, no liver tumors have ever been observed. Therefore, for greater sensitivity, a mouse model was used in which all mice would develop tumors. Different groups were tracked to provide survival times that could be compared statistically to determine if the treatments had an effect on the length of time between induction of C-MYC
expression in the liver and sacrifice due to tumor burden, a time frame defined in this study as “tumor latency.” We hypothesized that since ϕC31 integrase is associated with chromosomal aberrations in tissue culture, its expression might decrease tumor latency in these tumor-prone mice.
double transgenic, male mice were given doxycycline drinking water from conception until 7–8 weeks of age to suppress C-MYC expression in the liver during development, which would have been lethal () 
. Complete regulation of C-MYC expression by doxycycline was previously observed to have a time scale of four days 
. Therefore, at one week after initiation of sustained C-MYC induction, a hydrodynamic injection 
was administered containing no plasmid (saline only) or 20 µg each of a luciferase-expressing donor plasmid (pLiLucB) and plasmids containing vector backbone alone (pCS), expressing inactive integrase (pCSmI), or active wild-type integrase (pCSI) (). One group was given the active integrase plasmid pCSI alone (20 µg). The DNA dose of 20 µg per plasmid has been used to confer therapeutic levels of hFIX using ϕC31 integrase in mice 
. Two groups were not given hydrodynamic injection: one was given doxycycline drinking water for 7–8 weeks, and another was given doxycycline drinking water for one year. Mice that were injected with pLiLucB were imaged the next day to confirm high levels of luciferase expression in the liver, indicating a successful hydrodynamic injection. C-MYC induction preceded integrase expression because the integrase protein can only be detected by Western blot for up to one day after hydrodynamic injection 
Experimental design of tumorigenesis assay.
Mice were monitored weekly for tumor formation. The animals were sacrificed when it was expected that they would not have survived another week, as indicated by swelling in the upper abdomen () or signs of morbidity. Most of the mice with extensive hepatocellular carcinoma appeared behaviorally normal until the point of sacrifice. At autopsy, mice were dissected, photographed, and examined for the presence of liver tumors. Most tumors were multifocal, presumably arising from different tumor-forming cells (), as has been suggested previously 
. No differences in gross type, number, size, mass or distribution of tumors were observed between groups. Imaging was done every 2–3 weeks to monitor whether luciferase expression was observed in locations outside of the liver, indicating a possible luciferase-positive metastasis. Although several cases of metastasis were observed upon dissection (), none were detectable by luciferase imaging.
Treatment group and number of days from initiation of C-MYC overexpression until sacrifice (survival time) for each case of metastasis.
The seven groups of mice were observed for tumor formation to distinguish the effects of C-MYC induction, hydrodynamic injection, DNA administration, ϕC31 integrase protein, and ϕC31 integrase activity (). To make these effects easier to evaluate, we have separated the composite Kaplan-Meier survival curve () into several plots comparing these effects in a step-wise manner.
Survival curves suggest that hydrodynamic injection may contribute to C-MYC-induced tumor formation in the mouse liver.
Hydrodynamic delivery significantly decreased tumor latency
The survival times of mice in the control groups are compared in . The animals that did not receive C-MYC induction or any injections (yellow) survived until the end of the study, 400 days after 7–8 weeks of age. In contrast, induction of C-MYC expression in the liver beginning at adulthood (7–8 weeks) resulted in all of the mice being sacrificed prior to the end of the study with a median tumor latency of 154 days. These results confirmed that the LAP-tTA/TRE-MYC mouse model allowed for tight oncogene regulation.
Interestingly, in the group of mice that received a hydrodynamic injection of phosphate-buffered saline without any DNA present, the median tumor latency was only 105 days. There was one mouse that survived past 200 days that may have been an exception in some way, for example, by receiving an unsuccessful hydrodynamic injection. Similarly, a single long-surviving mouse was seen in other groups. To keep these outliers from having major effects on statistical significance, we chose the Gehan-Breslow-Wilcoxon statistical test to compare survival times, because this test gives less weight to later events. The group in which C-MYC
was turned “on” and no treatment was given (orange) was statistically different than the group that had C-MYC
“on” and received saline-only hydrodynamic injection (red; p
0.0359), indicating that there was a significant decrease in tumor latency associated with the hydrodynamic delivery method.
DNA delivery, luciferase expression, and imaging did not affect tumor latency
To test the effect on tumor latency of DNA without the integrase gene, we gave the transgenic mice hydrodynamic injections containing 20 µg each of pCS and pLiLucB (). Inclusion of this group was intended to control for both the integrase plasmid backbone elements as well as firefly luciferase expression and imaging every 2–3 weeks, which entailed injections of luciferin and anesthesia with isoflurane for a period of approximately 15 minutes. As shown in , the saline-only (red) and pCS/pLiLucB (purple) groups had nearly identical survival curves, except for one late survivor in the saline-only group. The pCS/pLiLucB group was statistically significantly different than the MYC “on”, no injection group (p
0.014), again implicating effects of the hydrodynamic injection.
Integrase expression resulted in similar tumor latency to that of untreated mice
To evaluate the effect of ϕC31 integrase protein expression independent of integration activity, we injected the plasmids pCSmI and pLiLucB () into a group of C-MYC expressing mice (, cyan). The pCSmI plasmid has a S20F mutation in the catalytic serine, rendering the ϕC31 integrase made by this plasmid unable to recombine DNA. We observed a statistically significant increase in tumor latency when inactive integrase protein was present, compared to the saline-only group (p
0.045). Again, note that we observed a very late surviving mouse in this group, which lived about twice as long as the second-longest surviving mouse in the group.
When the pCSI construct encoding active integrase () was administered with the pLiLucB donor plasmid (blue, ), the tumor latency also increased compared to the saline only group. According to the Gehan-Breslow-Wilcoxon test of statistical significance, the pCSmI/pLiLucB and pCSI/pLiLucB groups were not significantly different. To test if recombination of plasmid DNA was necessary for the observed survival benefit, we also administered 20 µg of pCSI without any attB donor plasmid (green) to a cohort of TRE-MYC/LAP-tTA mice. Presence of the attB-containing plasmid appeared to have no effect on survival ().
To summarize, the C-MYC “on”, pCSI/pLiLucB group was not statistically different than the C-MYC “on”, no injection group (), indicating that the presence of integrase appeared to counteract the tumor acceleration due to hydrodynamic injection. Hydrodynamic injection without integrase expression yielded a survival curve that was significantly different than the uninjected and pCSI/pLiLucB groups. The acceleration of tumor formation in mice that received a hydrodynamic injection appeared to be somehow abrogated by expression of the integrase. All groups are graphed together in on a longer x-axis.
Tumors did not have luciferase activity or ϕC31 integrase-mediated integration events
In order to investigate further whether ϕC31 integrase played any role in tumor formation, we analyzed tumors isolated from mice in the pCSI/pLiLucB group. We tested protein extracts from eight tumors and one metastasis from four mice in this group and found that none of them were positive for luciferase activity (). Additionally, we were unable to detect the luciferase gene by PCR in the six tumors from three mice in the pCSI/pLiLucB group that were tested (). This PCR would detect integration at any location in the genome. The tumors that tested negative for luciferase activity included the six tumors that tested negative for the presence of the luciferase gene, suggesting that the luciferase donor plasmid was not integrated and silenced in these tumors. To determine further if ϕC31 integrase mediated integration of plasmid DNA into the cells that generated tumors, we tested all dissected tumors for integration into the dominant pseudo attP
site in the mouse liver genome known as mpsL1 
, using a nested PCR. For each round, one primer was in the mouse genome at mpsL1 while the other primer was the in attB
sequence in the donor plasmid. PCR analysis was performed on DNA isolated from 19 tumors from 9 mice, including 7 tumors and 1 metastasis that were also tested for luciferase activity and six tumors also tested by PCR for the presence of the luciferase gene. No mpsL1 integration was detected in any tumors. Integration was detected only in DNA isolated from the normal-appearing part of the liver (). A GAPDH control demonstrated that a sufficient amount (200 ng) of genomic DNA was added to each PCR reaction. Thus, no evidence for ϕC31 integrase activity was found in any of the tumors from mice in the pCSI/pLiLucB group, suggesting that ϕC31 integrase may not have played a role in tumor formation in the LAP-tTA/TRE-MYC
transgenic mouse model.
Luciferase activity and PCR analysis of tumors from mice in the pCSI/pLiLucB group provide no evidence of ϕC31 integrase activity.
Luciferase imaging data and correlation to tumor latency
Mice were imaged on day 1 and every two weeks thereafter for luciferase expression for the first 14 weeks, followed by every three weeks thereafter. We have shown the luciferase imaging data to week 10 (), because after week 10 (day 70) animals began to be sacrificed, thus complicating the data with increasing statistical error as the group sizes decreased. Averaged luciferase values were normalized to the day 1 luciferase value to remove variability on account of transfection efficiency. The standard error was calculated using propagation of errors to take this normalization into account. pCSI/pLiLucB gave significantly higher long-term expression than pCSmI/pLiLucB (Student's t-test, p
0.014), demonstrating that ϕC31 integrase was active in the mouse liver. The pCS/pLiLucB and pCSmI/pLiLucB groups would still retain some luciferase expression due to random integration of the pLiLucB plasmid. The pCS/pLiLucB group maintained luciferase values that were significantly higher than pCSmI/pLiLucB (Student's t-test, p
). It is unclear why these groups have different long-term luciferase levels. One could speculate that since the pCS/pLiLucB group developed tumors faster than the groups given integrase-expressing plasmids, increased numbers of luciferase-positive cells in the liver cause higher long-term luciferase levels. We do not believe that luciferase is an ideal readout for overall levels of transgene expression in the liver, because the levels detected are dependent on the distance from the surface of the animal. In this animal model, tumors formed that may have complicated interpretation of the luciferase levels by displacing the normal liver away from the surface of the animal. It was the possibility of finding metastases that motivated our use of the luciferase transgene in the liver. However, luciferase imaging did not detect any luciferase-positive metastases ().
Luciferase expression and the relationship between initial expression values and long-term survival.
In order to correlate the efficiency of hydrodynamic injection with tumor latency, the luciferase expression on day 1 was plotted against survival time (). An R-squared value exceeding 0.95 would have indicated a trend correlating transfection efficiency and survival. The R-squared values for all groups were lower than 0.95, regardless of the method used to calculate the trend line, suggesting that variations in transfection efficiency may not have affected tumor latency.