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https://www.abbs.info ISSN 0582-9879 |
Conditional
Gene Activation in Cultured Hepatocytes Using a Ligand-dependent Chimeric
Cre Recombinase
ZHU Huan-Zhang1,2,3,
CAO Ying-Ying2, CHENG Guo-Xiang2, XUE
Jing-Lun1*
(1State Key Laboratory of
Genetic Engineering, Institute of Genetic, School of Life Sciences, Fudan
University, Shanghai 200433, China; 2Shanghai Transgenic Research Center,
Shanghai 201203, China; 3Institute of Pathology, Southwest Hospital, Third
Military Medical University, Chongqing 400038, China)
Abstract By combining liver-specific promoter and a chimeric
Cre recombinase, conditional gene activation could be finely achieved in hepatocytes
at selected time points. To this end, the expression vector of Cre-ERt under
the control of the mouse albumin gene promoter/enhancer, alb-Cre-ERt, was
constructed, and transfected into engineering BRL (Rat hepatocytes) and BRK
(Rat kidney) reporter cells which carries a chromosomally integrated ‘floxed’
βgeo gene, which is inserted between the promoter and the human alkaline phosphatase(
hAP) reporter gene, thereby preventing hAP reporter gene transcription, respectively.
After treated with 1 μmol/L 4-hydroxytamoxifen(4-OHT), a proportion of hAP
staining positive cells were detected by hAP staining. It was further confirmed
that ‘floxed’ βgeo cassette was removed by Cre excision by using PCR analysis
of cellular DNA. No background recombinase activity could be detected in the
absence of 4-OHT. Moreover, no hAP-positive cells could be detected in BHK
cells untreated or treated with 4-OHT. These data suggested that alb-Cre-ERt
expression vector was constructed successfully, and 4-OHT could induce Cre-mediated
recombination only in hepatocytes expressing Cre-ERt, thereby activating a
stably integrated hAP reporter gene. This provides a further foundation for
producing transgenic mice expressing such an 4-OHT inducible Cre recombinase
specifically in mouse liver.
Key words
Cre recombinase; inducible gene expression; hepatocytes;
alkaline phosphatase; lacZ
Liver is a potentially good target for somatic gene transfer because it plays
an important role in metabolism, and hepatocytes can deliver diffusible factors
into the circulatory system. This can help correct metabolic disorders, particularly
deficiency of liver-derived protein products. The ability to create defined
genetic modifications in hepatocytes provides a powerful strategy to understand
relevant functions of numerous hepatic genes in health and disease. Ligand-dependent
chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements
in cultured cells, such as F9 embryonal carcinoma cells[1, 2] and ES cells[3,
4], and mice[5－10]. The chimeric Cre recombinases are activated by 4-hydroxytamoxifen(4-OHT)
but not by endogenous estrogens because of specific point mutations in the
ligand-binding domains (LBD). By combining the use of liver-specific promoter
and the chimeric Cre recombinase, gene activation could be finely achieved
in hepatocytes at selected time points. To this end, we constructed the fusion
gene of Cre and the LBD of a mutated human estrogens receptor (ERt) that recognizes
the anti-estrogen 4-OHT, Cre-ERt, which under the control of the mouse albumin
gene promoter/enhancer(alb e/p) that was specifically active in hepatocytes.
The recombinant expression vector was transfected into the hepatocytes lines
conditionally expressing the reporter human alkaline phosophatase (hAP) gene,
it is shown here that in hepatocytes expressing such fusion proteins, 4-OHT
can efficiently induce Cre-mediated recombination, thereby activating a stably
integrated hAP reporter gene.
1 Materials and Methods
1.1 Construction of recombination plasmids
Small scale plasmid DNA preparation was performed according to Sambrook et
al.[11]; large scale preparation of plasmid DNA was done with the QIAquick
plasmid extraction kit (Qiagen). Restriction endonucleases and T4 DNA ligase
were purchased from New England Biolabs(NEB). Plasmids were grown in Escherichia
coli strain DH5α. Plasmid constructs were generated using standard cloning
techniques as described below. To generate palb-Cre-ERt expression vector
[Fig.1(A)], a 0.6 kb SmaI-SacII(blunted) fragment containing
CMV promoter from plasmid pCMV Cre-ERt (kindly provided by Prof. Chambon P)
was replaced with a 2.3 kb SacII(blunted)-EcoRV fragment containing
albumin enhancer and promoter(alb e/p)from p2335A(kindly provided by Prof.
Palmiter). The identity of construct was confirmed by restriction enzyme mapping
and DNA sequence analysis.
ZAP double reporter vector was kindly provided by Prof. Nagy. ZAP vector
is depicted as follow [Fig.1(B)]. The pCCAG promoter, comprising the CMV enhancer
and chicken β-actin promoter, drives expression of the reporter genes. The
first reporter, βgeo (lacZ / neoR�qfusion),
followed by three copies of the SV40 polyadenylation signal is flanked
by loxP sites and is removed by Cre excision. Use of the βgeo
provided not only a visual reporter but also a drug selection marker for cells
carrying the expression construct. The second reporter, expressed only after
Cre excision of the βgeo, is the hAP, followed by the rabbit β-globin
polyadenylationsequence. The identity of ZAP vector constructs was confirmed
by restriction enzyme mapping.
Fig.1 The alb-Cre-ERt and ZAP expression construct are depicted
(A) Structure of the alb-Cre-ERt expression vector. The DNA fragments contained
albumin gene promoter/enhancer(alb e/p), the rabbit β-globin intron, the fusion
gene of Cre-ERt, and the polyadenylation site (pA). (B) Structure of the ZAP
expression vector before and after Cre-mediated excision. The pCCAG promoter,
comprising the CMV enhancer and chicken β-actin promoter, drives expression
of the reporter genes. The first reporter, βgeo, followed by three
copies of the SV40 polyadenylation signal is flanked by loxP
sites and is removed by Cre excision. The second reporter, expressed only
after 4-OHT-induced Cre recombinase mediated excision, is hAP gene, followed
by the rabbit β-globin polyadenylation sequence.
1.2 Cell culture and manipulation
Rat hepatocytes (BRL-GNR1, type culture collection of Chinese Academy of Sciences)
and kidney cells(BRK-GNHa2, type culture collection of Chinese Academy of
Sciences) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with
10% fetal calf serum (Gibco), 2 mmol/L l-glutamine(Gibco), 1 u/ml penicillin
and 1 mg/L streptomycin at 37 °C in a humidity-saturated 5% CO
atmosphere. Cells were trypsinized, washed in PBS (137 mmol/L NaCl, 2.7 mmol/L
KCl, 4.3 mmol/L Na
and resuspended in PBS at 107 cells/ml. Then, 500 μl cell suspension
were mixed with 5 μg SfiI-ScaI linearized ZAP plasmid in 0.4
cm electrode gap cuvettes and electroporated at 250 V, 500 μF using a BioRad
Gene Pulser. Electroporated cells were plated into two 10 cm dishes, incubated
for 24 h, and then subjected to neomycin selection (G418, 200 mg/L) for 14
days. Colonies were mixed, propagated, and frozen, and aliquots were used
for lacZ staining, and then for PCR assay to confirm these cells stably
expressing the lacZ report gene.
Thawing 1000 μl cell by identificated suspension were mixed with 5 μg palb-Cre-ERt
plasmid in 0.4 cm electrode gap cuvettes and electroporated at 250 V, 500
μF using a BioRad Gene Pulser. Electroporated cells were plated into four
10 cm dishes, and incubated for 24 h, and treated with 4-OHT (Sigma). 4-OHT
was dissolved in 100% ethanol at 1 mmol/L; the final concentration was 1 μmol/L
(the final concentration of ethanol in the culture dishes was 0.01%). Cells
were used for X-gal and hAP staining, and analyzed by PCR after 24, 48, 72,
96 h exposure to 4-OHT, respectively.
1.3 X-gal staining
Cells were fixed in PBS containing 1% formaldehyde, 0.2% glutaraldehyde (Sigma).
lacZ expression was detected by in situ X-gal staining. Cells
were incubated overnight in PBS with 5 mmol/L potassium ferricyanide (Sigma),
5 mmol/L potassium ferrocyanide (Sigma), 2 mmol/L MgCl
X-gal (Sangon).
1.4 hAP staining of cells
For hAP staining, cells samples were rinsed in PBS and then endogenous alkaline
phosphatases were heat inactivated by incubation in PBS at 70 °C to 75 °C
for 30 min. Samples were rinsed in PBS, washed in alkaline phosphatase buffer
(100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, 10 mmol/L MgCl
for 10 min, and stained with purple alkaline phosphatase substrate NBT/BCIP
stain (100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, 50 mmol/L MgCl
0.01% sodium deoxycholate), 0.02% NP-40, 337 g/ml NBT (nitroblue tetrazolium
salt; Sangon), and 175 g/ml BCIP (5-bromo-4-chloro-3-indolyl phosphate, toluidinium
salt; Sangon) at 4 °C for 0.5 to 36 h. When staining was complete, the samples
were washed extensively in PBS containing 0.1% Tween 20 and 2 mmol/L MgCl
For X-gal/hAP double staining, plates were first stained for lacZ activity
and washed. Endogenous alkaline phosphatases were heat inactivated, and the
plates were stained for alkaline phosphatase activity. The number of hAP staining
cells were counted in samples of 10 000 cells in each experimental condition.
At least four fields per plate were analyzed.
1.5 PCR analysis
Cells were lysed with 1% SDS in 20 mmol/L Tris-HCl, pH 8, 50 mmol/L EDTA and
incubated for 16 h at 37 °C in the presence of 200 mg/L proteinase K (SABC,
Co.). Genomic DNA was isolated from the lysate by phenol extraction. To determine
whether the ZAP report cells stably express the lacZ report gene, the
primers were designed on lacZ gene sequence and synthesized by Sangon
Company. The sequence of primer for the PCR were as follows: the 5′ primer
for lacZ was 5′-ttc act ggc cgt cgt ttt aca acg tcg tga-3′ and the
3′ primer for lacZ was 5′-atg tga gcg agt aac aac ccg tcg gat tct-3′.
The PCR reaction condition contained 2 μl of 10×PCR buffer, 0.4 μl of 25 mmol/L
Mg2+, 1 μl of 15 mmol/L dNTP, 1 μl of 10 pmol/L each of primer,
2 u of Taq DNA polymerase, 1 μg sample DNA. A 20 μl total reaction
volume was obtained by adding sterile water, the reaction procedure of PCR
was: beginning 5 min at 95 °C, then 30 cycles with 94 °C denature for 30 s,
annealing and extension at 72 °C for 120 s; final extension at 72 °C for 10
min. Prepared a 1.8% agarose gel and loaded 10 μl PCR products in the gel
then electrophorese at 80 V for 40 min, observed the result of electrophoresis.
To confirm that floxed-βgeo cassette was removed by Cre excision, the sense
primer located in chicken promoter of ZAP vector was 5′-tgg taa tcg tgc gag
agg-3′ and the reverse primer located in the hAP gene was 5′-ccc agg aag atg
atg agg-3′. The PCR reaction condition was described as above. The size of
the fragment amplified from the non-recombined transgene is 5.5 kb, whereas
the recombined molecule, after excision of the Cre-ERt sequence, gives a 790
bp band.
2 Results
2.1 Construction of recombinant vector
The SmaI-SacII (blunted) fragment containing Cre-ERt
of plasmid pCMV-Cre-ERt was isolated, the SacII(blunted)-EcoRV
fragment containing albumin enhancer and promter (alb e/p) of p2335A was isolated,
respectively. The two individual fragments were ligated to generate palb-Cre-ERt.
The identity of palb-Cre-ERt was confirmed by restriction enzyme mapping
[Fig.2(A)] and DNA sequence analysis (data not shown), indicating that the
albumin enhancer and promoter were correctly inserted and linked. ZAP vector
was digested with HindIII-EcoRV, indicating that the DNA fragment
size was the same as anticipation(data not shown).
2.2 Generation of ZAP cells stably expressing the lacZ reporter
gene
To determine whether the Cre recombinase are able to regulate the expression
of a transgene in response to 4-OHT, ZAP report cells stably expressing the
lacZ report gene were generated. In brief, BRL cells and BHK cells
were first electroporated with ZAP vector, and selected for neomycin resistance,
respectively. After clones mixed and propagated, almost all cells displayed
strong LacZ staining(data not shown). BRL cells with ZAP vector or BHK cells
with ZAP vector were referred as BRL-ZAP cells or BHK-ZAP cells, respectively.
It was further confirmed that ZAP report cells stably expressing the lacZ
report gene were generated by PCR analysis of cellular DNA [Fig.2(B)].
Fig.2 Identification of palb-Cre-ERt recombinant and PCR analysis of genomic
DNA from BRL-ZAP cells stably expressing the lacZ reporter gene
(A) Identification of palb-Cre-ERt recombinant. 1, recombinant-HindIII;
2, recombinant-ScaI; 3, recombinant-EcoRV; 4, Vector-HindIII;
M, GeneRuler 1 kb DNA Laddar. (B) PCR analysis of genomic DNA extracted from
BRL cells transfected with ZAP expression vector. 1, DNA from BRL-ZAP cells;
2, positive control; 3, blank control; 4, negative control; M, GeneRuler 1
kb DNA laddar.
2.3 hAP gene is switched on by 4-OHT in hepatocytes
We next wanted to determine whether expression of hAP could be activated by
4-OHT specifically in hepatocytes. BRL-ZAP cells and BRK-ZAP cells were first
transfected with plasmid Alb-Cre-ERt, respectively, and then were treated
with 1 μmol/L 4-OHT. In BRL-ZAP cells treated with 4-OHT, a proportion of
hAP staining positive cells was observed (Fig.3), the X-gal and hAP
staining positive cells indicate the Cre recombinases preexcision and postexcision
status (Fig.3), respectively. A time-course experiment [Fig.4(A)] revealed
that the recombinase activity was rapidly induced by 4-OHT and reached a plateau
between 48 and 72 h, 38% of the BRL-ZAP cells were shown to activate hAP
expression by hAP staining. It was further confirmed that floxed-βgeo
cassette was removed by Cre excision by PCR analysis of cellular DNA [Fig.4(B)].
No hAP staining positive cells could be detected in BRL-ZAP cells untreated
with 4-OHT, indicating that hAP expression is repressed fully in the absence
of 4-OHT. Moreover, no hAP-positive cells could be detected in BHK-ZAP
cells untreated or treated with 4-OHT. It shows that the mouse albumin gene
promoter/enhancer is specifically active in hepatocytes.
Fig.3 Conditional expression of hAP mediated by 4-OHT-induced Cre recombinase
(A, B) Histochemical analysis of hAP expression by hAP staining in BRL-ZAP
cells treated with ethanol (A) or treated with 4-OHT (B). (C, D) Histochemical
analysis of hAP expression by X-gal and hAP double staining in BRL-ZAP cells
treated with ethanol (C) or treated with 4-OHT (D). A proportion of hAP staining
positive cells was observed only in BRL-ZAP cells treated with 4-OHT (B, D).
Fig.4 Time course of hAP expression and PCR analysis of Cre-mediated recombination
in BRL-ZAP cells treated with 4-OHT
(A) Time course of hAP expression mediated by 4-OHT-induced Cre recombinase
in BRL-ZAP cells. (B) PCR analysis of genomic DNA extracted from BRL-ZAP cells
transfected with Alb-Cre-ERt in the presence of 4-OHT, or ethanol, respectively.
A fragment of 790 bp is obtained after recombination only in the presence
of 4-OHT; M, GeneRuler 1 kb DNA laddar.
3 Discussion
The ability to manage the expression of genes introduced into mammalian cells
and animal would further progress in many areas of biology and medicine. For
instance, methods that allow the intentional manipulation of gene expression
would facilitate the analysis of genes whose production cannot be tolerated
constitutively or at certain stages of development. They would also be valuable
for clinical applications such as gene therapy protocols, where the expression
of a therapeutic gene must be regulated in accordance with the needs of the
patient. Presently, an efficient regulatory system allowing a tight control
of gene expression in mammalian cells has been developed. The system is based
on the bacteriophage P1 Cre recombinase, which efficiently catalyzes recombination
between two 34-bp loxP recognition sites, in the absence any cofactors[12,
13]. Depending on the relative orientation of the loxP sites, Cre-mediatedrecombination
results in DNA inversion, excision, integration, and translocation. This system
has been widely applied to the conditional gene activation or inactivation
strategies in mammalian systems, in cell lines and in transgenic mice[14,
15].
The conditional gene activation is that the Cre’s DNA excising capability
can be used to turn on a foreign gene by deleting DNA fragment that have been
flanked by directly repeated loxP sites (so called floxed) between
the promoter and the coding region of the transgene. Spatially or temporally
controlled gene expression can be obtained by placing the Cre gene under the
control of either a cell-specific or an inducible promoter, respectively.
More recently, a strategy based on the regulation of Cre activity, rather
than expression, has been described[1]. This strategy relies on LBD from steroid
receptors onto the Cre, thereby creating recombinases that require a ligand
for activity. At present, chimeric Cre recombinases that are selectively activated
by synthetic drugs provide useful tools to control the time of recombination.
To established a system that conditional gene expression only in hepatocytes
by a tamoxifen-inducible form of Cre recombinase, we constructed Alb-Cre-ERt
expression vector by placing the fusion gene of Cre-ERt recombinase under
the control of the liver tissue-specific promoter. Alb-Cre-ERt vector DNA
were transfected into BRL-ZAP reporter cells which carries a chromosomally
integrated ‘floxed’ βgeo gene, which is inserted between the promoter and
the hAP reporter gene, thereby preventing hAP reporter gene transcription.
In BRL-ZAP reporter cells expressing Cre-ERt, transcription of the hAP reporter
gene is repressed efficiently in the absence of 4-OHT, and is activated in
the presence of 1 μmol/L 4-OHT.
In this study, background recombination was not detected by hAP staining
and PCR assay in the absence of 4-OHT. The reason for this may be that first,
the insertion of three tandemly repeated sites for transcription termination
in ZAP vector is likely to prevent leakiness of hAP expression before 4-OHT
treatment. Second, as the recombinase is expressed in 38% of transiently transfected
cells, the background activity of Cre-ERt may be below the detection limit.
No background recombination was also reported in transiently transfected F9
cells and in a transgenic mouse line expressing Cre-ERt[1, 16]. However, very
low background recombination was reported in ES cells stably expressing a
recombinase similar to Cre-ER(GR)[17]. As suggested by Zhang et al.[16],
the basal activity could be due to proteolysis of the Cre-LBD fusion proteins
to yield constitutively active products in the cells.
In this study, the hAP expression cells after 4-OHT treatment were detected
by hAP staining, but only 38% frequency. The result is consistent with what
Sauer et al. report[12]. The current method of expressing the recombinase
in mammalian cells employed either DNA transformation or electroporation,
so sufficient Cre expression was achieved in only a small fraction of the
cultured cell population. Presently, using an adenovirus (Ad) expression vector,
a drug-selection strategy, or protein transduction technology, the desired
cell population were obtained[18, 19]. If a sufficient level of the functional
recombines is obtained simultaneously in 100% of the cultured cells, the recombinase-mediated
gene manipulation in mammalian cells can have much wider application.
To sum up, we successfully constructed vector expressing a tamoxifen-inducible
Cre-ERt fusion protein specifically in hepatocytes, demonstrated that tamoxifen
can induce Cre-mediated recombination, thereby activating a stably integrated
hAP reporter gene. Transgenic mice expressing such an 4-OHT-inducible Cre
recombinase may thus provide a new and useful genetic tool to create defined
genetic modifications in liver at specific times during development or in
adult animals. ZAP cells line with double reporter gene provided a convenient
and reliable reporter system for Cre-mediated recombination.
Acknowledgements We thank Dr. Chambon P of Universite Louis Pasteur
for providing plasmid pCMV Cre-ERt, Dr. Nagy A of University of Toronto for
plasmid pZAP, and Dr. Palmiter for plasmid p2335A.
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Received: December 20, 2002Accepted: February 21, 2003
This work was supported by grants from the National Natural Science Foundation
of China （No.30070380, No.30170145）
*Corresponding author: Tel, 86-21-65642424; e-mail, [email protected]