Highly active gene expression driven by the adenoviral MLP and TPL sequence, observed during late stages of adenovirus infection, has led to their in vivo use for gene expression . However, to achieve even higher levels of gene expression, use of the CMVie promoter/enhancer has been preferred for both in vivo and in vitro studies and applications [14,26,28,37].
Part of the observed difference in expression levels between these two systems may be explained by the roles they fulfill in their endogenous viral contexts. Namely, the adenovirus MLP, which facilitates expression of all late genes, is only active during the end stages of adenovirus replication and depends on transactivation by early viral products [30,51]. However, the CMVie promoter/enhancer drives expression of the very first genes required for cytomegalovirus replication and, therefore, is not dependent on the presence of other viral components for activity [39,41].
In this study, we tested whether providing additional adenovirus sequences, in trans, could enhance MLP-TPL activity to that observed for CMVie, using a GFP reporter gene and assaying for transcript and protein levels. For this purpose we made use of the adenovirus dl309, which has the same properties as the wild-type adenovirus serotype 5 with the exception of the E3 region [4,20,21]. CHO cells were chosen as the cellular background for this study because they are the most widely used mammalian cell line for recombinant protein production .
Our data confirmed that outside of their endogenous viral contexts, activity of CMVie was significantly higher than that of MLP/TPL for transgene expression. Although GFP expression driven by CMVie was higher at both transcriptional and protein levels, relative to MLP/TPL, this effect was more pronounced in the former. This was expected, since the TPL facilitates late mRNA transport and accumulation in the cytoplasm and is responsible for the selective translation of the late viral proteins instead of the cellular proteins [3,24,49]. Adenovirus E1B 55k and E4 orf6 play the main role in the Forskolin of viral late mRNA, containing the TPL, from the nucleus to the cytoplasm [2,8,17,23,32,31,42], while cellular mRNA transport is blocked by the same complex . In addition, the TPL allows ribosome shunting, in a similar manner to the internal ribosome entry site (IRES) recognition, which avoids the scanning mechanism that is needed for cellular mRNAs and the required cap-binding [47,48].
GFP expression driven by the MLP/TPL was higher upon adenovirus infection, relative to non-infected cells. This enhancement was due to the expression of the E1A 12S and 13S protein products which stimulate cell division and growth . They play an important role in activating the expression of adenoviral E2 proteins and other cellular S-phase proteins . However, even with the added activity stimulated by adenovirus infection, reporter gene expression from the MLP/TPL construct did not reach levels observed for CMVie in non-infected and Ad5dl309 infected cells. Interestingly, the CMVie promoter/enhancer also showed enhanced expression in adenovirus-infected cells, suggesting that adenoviral components, provided in trans, can stimulate activity of this regulatory sequence, most likely in a similar manner to that observed for MLP. In addition, the effects of adenovirus infection on reporter gene expression from both systems were greater on transcription than on translation levels, although both increased relative to non-infected control. As more information about new transcripts generated by alternative splicing from the adenoviral genome is being uncovered [36,50]. That might reveal some specific sequences and/or proteins that can be tested to enhance transgene expression from the MLP-TPL.
Mastitis is the most expensive problem facing the dairy industrial communities. Globally, the estimated economic losses due to mastitis reached about 533billion$ ; the estimated economic loss of milk per cow per one lactation cycle due to mastitis is 70%, while it was 14% due to premature culling; on the other hand, it was 7% due to the exclusion of the mastitis milk and finally it was 8% due to the cost of the veterinary medication, of the total losses reported worldwide [2–4]. Staphylococcus aureus (S. aureus) is considered one of the main worldwide causative agents of 40–70% of contagious bovine mastitis [2,3]. Moreover, S. aureus is known worldwide as a toxigenic food-borne bacteria which is considered a dangerous threat to human life, because if S. aureus counts reached inside the food like milk to 105–106CFU/ml or gm at temperatures between 10°C and 46°C, then it can be able to secrete dangerous heat-stable enterotoxins . Furthermore, S. aureus has the ability to convert to a multi-drug resistant S. aureus knowing worldwide as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) [6,7]. MRSA is known worldwide as a multi-drug resistance acquired hospital pathogen, but recent reports revealed that MRSA was associated with cases of bovine mastitis . Vancomycin was considered the drug of choice to overcome MRSA infection, but recently in 1997, MRSA that becomes intermediate susceptibility or resistant to vancomycin (VRSA) was begun to appear, meaning that MRSA can be a vancomycin resistance (MRSA+VRSA) . The emergence of MRSA and VRSA in the cases of mastitis and its return harmful effect on the human being, with increasing failure in their treatment, and the associated high morbidity and mortality within both human and animals, all of that, raised a necessity to experimentally searching for a new therapeutic anti-MRSA and VRSA agents.