Cullin 4A (Cul4A) belongs to the family of evolutionarily conserved cullin proteins, including seven related ones (Cul1, 2, 3, 4A, 4B, 5, and 7) in humans. Cul4A forms a part of the multifunctional ubiquitin-protein ligase E3 complex by interacting with ring finger protein (ROC1) and damaged DNA binding protein (DDB1). Through ubiquitin-mediated proteolysis, Cul4A regulates many critical processes in cells. Cul4A
is a critical gene for hematopoietic cell survival and development. Overexpression of Cul4A
reportedly increases cell growth and causes the disruption of the G2–M cell cycle in irradiated mammary epithelial cells. Previous studies (Kopanja et al., 2009
) showed that Cul4A
is amplified in breast and liver cancers, is implicated in the ubiquitination and proteolysis of tumor suppressors such as p53, and may contribute to tumorigenesis and cancer development through ubiquitination and then proteolysis of tumor suppressors.
gene restricts cellular repair capacity (Angers et al., 2006
; Li et al., 2010
). It does so by orchestrating the concerted actions of nucleotide excision repair (NER) and the DNA damage-responsive G1/S checkpoint through selective degradation of the DDB2 and XPC DNA damage sensors and the p21/CIP1/WAF1 checkpoint effector (Liu et al., 2009
). We previously found that Cul4A
is an oncogene for mesothelioma (Hung et al., 2009
). However, the relationship between Cul4A and the occurrence and metastasis of other tumors such as mesothelioma and lung cancer is seldom reported, and the potential oncogenic role of Cul4A has been little studied.
Mouse models have been instrumental in developing our knowledge of signaling pathways in vivo
and of the aberrations in these pathways that are causally associated with disease. Studies of the Cul4A
gene in mice have been restricted to the loss-of-function studies using Cul4A
knockout mice (Liu et al., 2009
). For instance, it has been shown that Cul4A
knockout mice are resistant to ultraviolet (UV)-induced skin carcinogenesis and that Cul4A restricted cellular repair capacity through selective degradation of p21, DDB2, and XPC (Liu et al., 2009
). However, gain-of-function studies of Cul4A
using transgenic mice have not been reported.
The relationship between Cul4A
and cancer is not well understood. Because a transgenic mouse model is needed to explore the potential oncogenic role of Cul4A
, we sought to generate one with lineage-tracing that conditionally overexpresses Cul4A protein on Cre-mediated recombination. In this mouse model, before Cre-mediated recombination, LacZ
was expressed in the majority of embryonic and adult tissues that were successfully transfected with the gene vector. On Cre-excision in selected organs, LacZ
expression was replaced by expression of Cul4A
and a Myc-tag
. In our model, Cre-excision was induced in mouse lungs by inhalation of an adenovirus vector encoding Cre recombinase (Ade-Cre) (DuPage et al., 2009
). To generate this mouse model, we used the pCALL2 expression vector, which has been previously reported (Ding et al., 2002
; Elghazi et al., 2008
; Lobe et al., 1999
; Novak et al., 2000
). In our study, a Myc-tagged full-length Cul4A
cDNA was subcloned downstream of the loxP-flanked LacZ/neoR
. Between the loxP sites, the LacZ
sequence is followed by a triple repeat of the SV40 polyadenylation signal (Zinyk et al., 1998
) to ensure a transcriptional stop.
Before the Cre-mediated excision, the promoter regulated expression of the loxP-flanked LacZ/neoR
. The Cre recombinase of the P1 bacteriophage catalyzes recombination between two specific 34-bp consensus sequences (loxP sites) with high specificity and efficiency (Sternberg and Hamilton, 1981
). The loxP sites are palindromic, except for an 8-bp asymmetric core sequence that provides each loxP site with an orientation (Hoess et al., 1986
). Introduction of Cre enzyme results in recombination between the loxP sites and excision of the intervening DNA sequence. Upon Cre-mediated excision, LacZ
expression is switched to the expression of the Cul4A
and a Myc-tag
as a monitor of expression (). The expression of Cul4A can be manipulated in an organ of interest by Cre-excision in that organ.
FIG. 1 pCALL2-Cul4A and the three positive lines. (a) Diagram of the pCALL2-Cul4A transgene construct before and after Cre-mediated excision of the floxed LacZ-neoR sequence. (b) Design of the PCR primers for genotyping. (c) PCR data (pc, positive control; nc, (more ...)
The pCALL2 vector constructed with the Cul4A
gene (We hereafter refer to the constructed vector as pCALL2-Cul4A
.) was used to generate transgenic mice using embryonic stem cell (ES)-based transgenesis. By standard methods (Nagy, 1997
), the pCALL2-Cul4A
was transfected into ES cells. After clone selection, the chosen clones of ES cells with pCALL2-Cul4A
were used to generate chimeric mice. Transgenic mice were mated with FVB/N mice to produce transgenic offspring. The pCALL2-Cul4A
transgene was genotyped by polymerase chain reaction (PCR) using genomic DNA isolated from the tail. We successfully obtained three chimeric mouse lines (lines 1, 6, and 23) from 24 lines (). Whole mount X-gal staining was positive in the tails () and the lungs () of these three mouse lines, but varied in strength. Line 6 exhibited weak LacZ
expression in the staining of tail and lung, whereas Lines 1 and 23 showed similar strong LacZ
Transgene expression was assessed by X-gal staining in the brain, heart, lung, stomach, gut, liver, pancreas, spleen, kidney, and muscle of transgenic mice from the chimeric mouse lines 1 and 23. These lines showed similar stronger LacZ (X-gal) expression in most organs when compared with negative controls (The X-gal staining data for lung, brain, stomach, gut, muscle, pancreas, and kidney are shown in . Data for heart, liver, and spleen are not shown.). However, LacZ expression varied in different organs and tissues. In the brain, LacZ expression was higher in the white matter than in the gray matter. In the lung, the bronchi and alveoli showed the same level of LacZ expression. Overall, LacZ expression was strongest in the gastrointestinal organs, especially in mucous membrane, although it was weak in the chorion and smooth muscle layers and the glands of mucous membrane in the gastrointestinal organs (). In the kidney, expression was higher in the medulla than in the cortex (). In the pancreas, LacZ expression was apparent, especially in the pancreas islets (). In the spleen, expression of LacZ was homogeneous (data not shown). Lower levels of LacZ expression were observed in the muscle () and heart (data not shown), and none was observed in the liver (data not shown). The fact that high levels of the transgene were expressed in the stomach, gut, lung, brain, spleen, and pancreas suggests that, in this mouse model, studies of Cul4A should be feasible in these organs. The lower levels of LacZ expression in the muscle and heart and the lack of expression in the liver suggest that studies of Cul4A would not be feasible in those organs.
FIG. 2 Transgene expression assessed by X-gal staining in several organs of transgenic mice from lines 1 and 23 (a–n), and immunofluorescence staining for anti-LacZ (X-gal) in mammary glands from Line 23 (o–t). In (a–n), the transgene (more ...)
We also did immunofluorescence staining for anti-LacZ (X-gal) in mammary glands. The data showed strong LacZ expression in the chimeric mouse lines 1 and 23 and a remarkable difference between control and pCALL2-Cul4A mouse lines 1 (data not shown) and 23 (). This result suggested that the pCALL2 vector highly expressed in mammary glands of the chimeric mouse lines and that studies of Cul4A should be feasible in mammary glands in this mouse model.
To assess Cul4A expression, Cre excision was induced in the lungs of transgenic mice. Half of the transgenic mice inhaled Ade-Cre. Simultaneously, the other half inhaled Ade-GFP without Cre. Six weeks later, lungs from these mice were dissected. To determine the expression of Cul4A and Myc-tag with or without Cre-excision, lung sections from pCALL2-Cul4A mouse lines 1, 6, and 23 with Ade-GFP or Ade-Cre were immunostained with anti-Cul4A and anti-Myc-tag antibodies, respectively. The lungs of mice with Ade-Cre were positive for both anti-Cul4A and anti-Myc-tag, whereas the lungs of mice without Cre-excision were negative for both. Interestingly, we noticed some potential hyperplasia regions in the mouse lungs after Ade-Cre inhalation (, the potential hyperplasia regions were marked by arrows.). Possibly, this is due to the oncogenic functions of the Cul4A protein. Further studies with longer induction periods will be needed to study these effects. Among the three lines, immunostaining was weakest in Line 6 (). This result was similar to that of the whole mount X-gal staining (). The lung sections from pCALL2-Cul4A mouse lines 1, 6, and 23 with Ade-GFP and Ade-Cre were also immunofluorescence stained for anti-Cul4A, anti-Myc-tag, and anti-LacZ. The results for Cul4A and Myc-tag were the same as those for immunostaining—the lungs of mice with Ade-Cre were positive, and the lungs of mice with Ade-GFP were negative. Immunofluorescence staining for LacZ was negative in lungs with Cre-excision and positive in lungs without Cre-excision ( for mouse line 23; data not shown for mouse lines 1 and 6). Taken together, all of these results confirmed that Cre-excision was successful. In mice that inhaled Ade-Cre, the LacZ gene was excised; therefore, these lungs stained positive for anti-Cul4A and anti-Myc-tag antibody and negative for anti-LacZ antibody. We used the same virus particle of Ade-Cre but with GFP (instead of Cre) as control. In mice that inhaled Ade-GFP, the pCALL2-Cul4A gene could not be expressed because the LacZ and the triple repeat of the SV40 sequences were not excised. Consequently, LacZ continued to be expressed instead of Cul4A and Myc-tag.
FIG. 3 Immunostaining in lung sections from pCALL2-Cul4A mice with Ade-GFP (a,e) or Ade-Cre (b–d, f–h). (a–d) Anti-Cul4A; (e–h) Anti-Myc-tag; (b,f) Line 6; (c,g) Line 1; and (d,h) Line 23. The potential hyperplasia regions were (more ...)
Immunofluorescence staining in lung sections from pCALL2-Cul4A mice with Ade-GFP (top panels) or Ade-Cre (bottom panels). (a) Anti-Cul4A. (b) Anti-Myc-tag. (c) Anti-LacZ. Scale bar, 100 μm.
In conclusion, we have produced a gain-of-function transgenic mouse model that overexpresses Cul4A in a Cre-dependent manner. We believe that these transgenic mice are valuable for further characterization of the Cul4A gene in different organs and at different developmental stages. The lineage tracing capabilities of our model are powerful tools to elucidate the importance of Cul4A in cell fate during development, as well as in the plasticity of mature cells. This mouse strain provides a new model for studying the Cul4A complex in vivo.