Practical Range of Effective Dose for Cre Recombinase‐Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells

Abstract

Recently, the importance of regulating transgeneexpression in mammalian cells has been well recognized in the fields of basic research and future gene therapy. Site-specific recombinases, like Cre (6,30,32) and FLP (2-4, 7, 24, 26, 44), are useful tools for this purpose. Cre recombinase derived from the bacteriophage P1, in particular, has been utilized for efficient gene activation and inactivation in mammalian cells (9,37). Cre is a 38-kDa protein that mediates the excisional deletion of a DNA sequence flanked by a pair of loxP sites, a Cre-specific recognition sequence of 34-base pairs, with the same orientation. The gene activation Abstract: The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR␣ promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.

Disruption of Foxg1 expression by knock-in of Cre recombinase: Effects on the development of the mouse telencephalon

“…In primary cultures of mouse embryonic fibroblasts, in the absence of exogenous loxP sites, the introduction of the cre gene via a viral vector resulted in recombination events, growth arrest and, often, increased cell death (Loonstra et al, 2001). Similar effects have been noted in a variety of mammalian cell lines (Pfeifer et al, 2001, Silver and Livingston, 2001, Baba et al, 2005. More detailed analysis indicated that proliferating, rather than postmitotic, cells were vulnerable to the effects of Cre recombinase, as cryptic recombination events were only observed in mitotically-active cells (Loonstra et al, 2001).…”

“…There have been two reports of overt pathology in cre-expressing transgenic mice, where high levels of Cre resulted in male infertility when expressed in postmeiotic spermatids (Schmidt et al, 2000) and hydrocephaly when expressed in neuronal progenitors (Forni et al, 2006). In the latter study, the genotoxic effects required the levels of Cre recombinase to reach a critical, high threshold in the cell nucleus, as had been reported previously in vitro (Baba et al, 2005). Thus, hydrocephalus was observed in homozygous, but not hemizygous, mice in a transgenic line, in which multiple copies of the cre transgene had been inserted into unknown locations in the genome (Forni et al, 2006).…”

“…An important difference between the two lines is the presence of a nuclear localization signal in the Foxg1-cre line (Hebert and McConnell, 2000), which is absent in the Emx1-cre line (Gorski et al, 2002), likely resulting in higher levels of Cre recombinase in the nucleus. As noted above, there is evidence in vivo and in vitro that the accumulation of Cre recombinase specifically in the nucleus, rather than the absolute levels in the cell, underlies the DNA damage and growth inhibition observed in the absence of exogenous loxP sites (Baba et al, 2005, Forni et al, 2006. Thus, because of the time required to achieve threshold levels of nuclear Cre recombinase, overt abnormalities may not be apparent until several days after cre is first expressed.…”

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

“…In later stages of osteogenic differentiation (in osteoblast-like cells), fluoride (fluoroaluminate) treatment leads to co-activation of FAK and its analog, proline-rich tyrosine kinase-2 (PYK2), and subsequent downstream stimulation of MAPKs including ERK 1/2 [43], implying that the two kinases may act cooperatively. However, siRNA knockdown of FAK in our cells results in no compensatory increase in PYK2 phosphorylation on Y402 and still leads to a reduction in ERK activity, suggesting that at this earlier stage of differentiation, at least, FAK appears to be acting alone.…”

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

“…An earlier study showed that CRE was very effective in T. brucei but could not overcome its toxicity [17]. Indeed, toxicity from excess CRE has been reported recently in many systems [18][19][20][21]48], and, considering the potency of the GPEET promoter driving CRE expression in procyclic T. brucei, some degree of toxicity would not be unexpected. Our goal was to implement a system in which CRE would excise from the genome a combined positive and negative marker by recognizing the flanking loxP elements at a safe level of CRE expression.…”