|
|
|
Original Paper
|
|
|||
|
Acta Biochim Biophys
Sin 2007, 39: 235�246 |
||||
|
doi:10.1111/j.1745-7270.2007.00258.x |
Tetracycline-inducible expression systems: new strategies and practices in the transgenic
mouse modeling
Yan SUN1,2, Xigu
CHEN1*,
and Dong XIAO2*
1 Center of Experimental
Animals, Sun Yat-Sen University, Guangzhou 510080, China;
2 Institute of
Comparative Medicine and Center of Experimental Animals, Southern Medical
University, Guangzhou 510515, China
Received: October
9, 2006�������
Accepted: December
11, 2006
This work was supported
by the grants from the National Natural Science Foundation of China (No.
30271177 and 39870676), the National 9th Five-year Program (No. 101033), the
Major Science and Technology Projects of Guangdong Province (No. B602), the
Natural Science Foundation of Guangdong Province (No. 021903), the Postdoctoral
Fellowship Foundation of China (Series 29), and the Special Fund of Scientific
Instrument Collaborative Share-net in Guangzhou (No. 2006176)
*Corresponding
authors:
Dong XIAO: Tel,
86-20-62789009; E-mail, [email protected]
Xigu
CHEN: Tel, 86-20-33151566; E-mail, [email protected]
Abstract������� To accurately analyze the function of transgene(s) of interest in transgenic mice, and to generate credible transgenic animal models for multifarious human diseases to precisely mimic human disease states, it is critical to tightly regulate gene expression in the animals in a conditional manner. The ability to turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedented flexibility in dissecting gene functions in health and disease. Pioneering studies in conditional transgene expression have brought about the development of a wide variety of controlled gene expression systems, which meet this criterion. Among them, the tetracycline-controlled expression systems (e.g. Tet-off system and Tet-on system) have been used extensively in vitro and in vivo. In recent years, some strategies derived from tetracycline-inducible system alone, as well as the combined use of Tet-based systems and Cre/lox P switching gene expression system, have been newly developed to allow more flexibility for exploring gene functions in health and disease, and produce credible transgenic animal models for various human diseases. In this review these newly developed strategies are discussed.
Key words������� tetracycline-inducible expression system; Cre/lox P
system; transgenic mouse modeling; leaky expression; stringent control;
lineage-specific gene expression; lineage-specific RNAi
The Human Genome Project has
revealed the sequence information (http://wit.integratedgenomics.com/GOLD)
of many of the genes that make us what we are. Therefore, a significant challenge
for scientists over the next few decades is to annotate the human genome with
functional information. This effort will enable us to gain a full understanding
of the molecular mechanisms and pathways underlying normal development, as well
as those responsible for pathogenesis.
One powerful approach is the
transgene over�expression of any given gene(s) in genetically engineered mouse
model to explore the role(s) of the gene(s) in vivo. Conditional
transgenic mice are becoming increasingly popular for precisely regulate gene
expression in a temporal and spatial pattern. The ideal controlled
over-expression system should permit the investigators to rapidly and
reversibly switch the transgene expression on and off, exclusively in the
desired cells or tissue(s) at any time point during development. Tetracycline
(Tet)-inducible expression system is one of the most prominent and
widely-accepted inducible systems so far. There are two basic variants of the
tetracycline-inducible expression system: the tTA (Tet-off) system [1] and the
rtTA (Tet-on) system [2]. These systems, especially Tet-on system, have been
most extensively employed in the transgenic mouse modeling [3-7].
The tetracycline-dependent
regulatory systems (Tet-based systems) have been successfully employed to
define the development-dependent and development-independent biological and
pathological processes [4-7].
These systems, however, are not always tight but leaky because of the inherent
defects in Tet-based systems and promoter leakiness [5,6,8-12]. In some cases, leak is not a big
problem; in some others, however, it is a real problem and must be avoided [9-13].
By integrating the
tetracycline-dependent expression systems with an Cre/lox P switching
gene expression system, four labs [14-17] have
already developed a unique transgenic mouse system based on (r)tTA dependent
doxycycline (Dox)-mediated activation of target gene(s) following a
Cre-excision event to achieve a temporal, spatial and lineage-specific gene
expression in mice. This unique and versatile system is very useful for
functional analyses within specific cells or tissue(s) with the added advantage
of temporal regulation of gene activity within daughter cells of a specific
lineage in an inducible manner.
This review will give a brief
introduction of the tetracycline-dependent expression systems and focus on the
newly developed or developing strategies and their applications in transgenic
mouse modeling.
Components and working principles of Tet-based systems
and Cre/lox P system
Tetracycline-inducible
expression systems
Together with a specific
promoter, the tetracycline-inducible systems permit relatively stringent,
reversible (on�off),
quantitative, temporal and spatial regulation of transgene expression in a wide
range of cells in culture [8], as well as in transgenic animals (http://www.zmg.uni-mainz.de/tetmouse)
[3-7]. Use of the Tet-based systems requires
two coordinate building blocks: the ligand-dependent transactivator (r)tTA as
the effector and a tetO-CMV minimal promoter cassette regulating the
expression of the transgene as the responder. Fig. 1(A) fully
demonstrates the underlying mechanisms of the Tet-on system in the transgenic
mouse modeling.
Cre/lox
P switching expression system
Currently, the most widely
used site-specific DNA recombination system in mice is Cre/lox P system
[18,19]. The Cre/lox P system has two components: Cre recombinase derived
from bacteriophage P1 and two 34-bp lox P sites that Cre recognizes; the
site-specific recombination is accomplished by Cre-mediated catalysis of
reciprocal recombination between the two lox P sites in both tissue
culture cells and mice [18,19]. The general strategy for conditional gene
expression mediated by the Cre/lox P switching expression system is to
excise the intervening transcription STOP cassette [Fig. 1(B)] or DNA
sequence flanked by two lox P sites placed in the same direction,
thereafter achieving conditional gene activation [Fig. 1(B)] or
inactivation [18,19]. The conditional regulation of transgene expression is
accomplished by operating Cre expression in a specific place and/or at a
specific time.
Optimal "off/on" regulation of transgene expression
accomplished by the improved Tet-based systems in the transgenic mouse
modeling
Leaky expression in Tet-based
systems compromises the expected tight regulation
If a gene is to be kept inactive
most of the time and turned on only occasionally, Tet-on system appears to be
more appropriate than Tet-off system; moreover, of these two systems, rtTA
system is more suitable for rapid induction of gene expression.
But unfortunately, leaky expression,
which is derived from both the inherent defects in Tet-based systems and the
promoter leakiness caused by promoter-dependent or integration site-dependent
effects, compromises the desired stringent regulation of transgene expression
[5,6,8-13]. In theory, the promoter
leakiness may be effectively avoided if large numbers of transgenic
microinjections are undertaken to make a great number of transgenic animals.
The inherent shortcomings are attributed to the fact that rtTA retains some
affinity for tetO sequences even without Dox and to the unwanted
residual activity of the tetO-CMV responder even when the active (r)tTA
is absent [Fig. 1(A)] [5,6,9-11].
This is evident from the undetectable or detectable expression levels of
transgene activation, and further phenotype induction in
animals or cells which are not receiving Dox [5,6,9-13].
Under most circumstances, the
leak in Tet-based systems is quite acceptable by the investigators who use
these systems. In some circumstances, however, the detectable and undetectable
levels of undesired transgene leaky expression in animals
greatly limits the use of Tet-based systems in precisely modeling the complex
biological or human disease processes or in evaluating the effects of gene
product(s).
For instance, if the
product(s) of the given transgene(s) is/are toxic or unwanted, even a low level
of expression could be detrimental to embryos, preventing any further analysis
of the potential phenotype during late antenatal development or in the infancy
and adult. Tet-off system was employed in the transgenic mouse modeling to
govern the regulated expression of the cell-autonomous, lethal diphtheria toxin
A (DTA) gene [20]. Data indicated that transgenic mice (responder mice), which
harbored the tetO-DTA target transgene, were generated at a tenfold
lower frequency compared with previous production [20].
The complete "off"
and "on" regulation derived from the tightly controlled gene
expression system will be very useful to express the antigen genes of hepatitis
virus in a temporally restricted fashion and precisely define the immunological
reactions against transgene products such as infectious agents and pathogenesis
of hepatitis. When the activation of target gene expression in the animals is
mediated by the Cre/lox P switching expression system, the transgenic
animal is immunocompetent for the transgene product(s). To examine the immune
response to hepatitis C virus (HCV) structural proteins and pathogenesis of
hepatitis C, Wakita et al. [21] used the Cre/lox P system to
express the core, E1 and E2 proteins efficiently and conditionally in
transgenic mice (e.g. CN2 mice), providing a useful "non-immune
tolerant" animal model with which to investigate the host immune response
against HCV infection and the pathogenesis of hepatitis C [21-24]. The hepatitis B virus (HBV) and HCV
transgenic animals derived from non-tight gene expression regulatory systems
(i.e., heavy metal-inducible MT-1 promoter with high basal activity in the
absence of induction [25-27], Tet-off system [12] and
Tet-on system [10]) and from constitutive gene expression systems [10] are not
immunocompetent for the transgene product(s), e.g. viral antigen(s), because
the viral antigen gene(s) begin(s) to express before the formation of immune
system of organism. In these "immune tolerant" mice, the immune
system can not recognize the xenobiotic nature of these viral antigen(s);
actually, the immune system plays rather important roles in the pathogenesis of
both hepatitis B and C [12,21-24]. Although Tet-based
systems permit relatively tight regulation of transgene expression and leaks
very slightly, the data demonstrated that the amount of leakage was not enough
to detect biochemically but sufficient to induce immune tolerance [12].
The phenotypes and
pathological analysis demonstrates that CN2 mice for HCV, but not "immune
tolerant" animal model for HBV or HCV can mimic disease states in human
precisely and truly. In "non-immune tolerant" CN2 mice, several days
after the transgene activation, the core, E1 and E2 proteins could be detected
in liver lysates with concurrent increases in serum alanine aminotransferase
levels, and the level of circulating core protein was primarily dependent on
hepatocyte destruction;
subsequently, seven days and fourteen days after
AxCANCre administration, the development of substantial hepatic pathology in
CN2 mice which was not present in the naive CN2 mice or CD41&CD81
cell-depleted CN2 mice was revealed in the liver and the anti-core antibody
response was detected, respectively [21-24].
The findings from CN2 mice suggest that HCV proteins are not cytopathic
directly and that the host immune response plays an important role in HCV
infection. Whereas in "immune tolerant" transgenic mice for HBV and
HCV, the expression of hepatitis virus antigen genes was detected in the liver,
but there have no significant differences between "immune tolerant"
transgenic mice and normal mice in serum ALT
and aspartate aminotransferase levels, the antibodies against virus
antigens in serum were not detected, and the normally pathological changes in
the liver could not be observed [12,25-31].
Collectively, the "immune tolerant" animal models for HBV or HCV
derived from non-stringent gene expression regulatory systems, which may not
allow for the development of an ideal disease model for human hepatitis, and
from the constitutive gene expression systems can not precisely and truly mimic
the states of human diseases, such as virosis hepatitis.
Although the
tetracycline-inducible systems with acceptable levels of leak have been used
successfully to define the development-dependent and -independent biological
and pathological processes, to some extent, the confounding effects of
transgene leak in Tet-based systems may interfere with analysis of gene
functions and phenotype in the transgenic mouse modeling. Combined tTS and rtTA
systems, but not rtTA system alone, can be used to define the natural process
of in vivo injury and repair responses accurately and truly [11,13]. The
incorporation of tTS into Tet-based transgenic system effectively decreased
basal transgene leak to undetectable levels and totally eliminated the
IL-13-induced phenotype without Dox administration [13]. Furthermore, in the
CC10-rtTA-IL-13 double transgenic mice, IL-13, mucus metaplasia, inflammation,
alveolar enlargement and enhanced lung volumes were noted at base line, and
increased greatly after Dox administration, whereas in the CC10-rtTA/tTS-IL-13
triple transgenic animals, IL-13 and the IL-13-induced phenotype could not be achieved
without Dox [13]. The combined use of tTS and rtTA systems in producing
transgenic mouse models with a truly regulatable "on/off" switch of
gene expression is very useful in studies in which the critical windows of
development and the natural process of injury and repair are defined precisely
and truly.
Taken together, although
Tet-based inducible transgenic modeling systems with acceptable levels of leak
have provided a great deal of valuable information on the functions of many
genes and on the processes and pathogenesis of some human diseases, under
certain conditions such "leaky" systems derived from intrinsic
defects of systems are not always tolerable for models of more complex human
diseases and also not suitable for more accurately answering the complex
questions. For example, whether the effects of transgene products are
development-dependent?
Uses of improved rtTA
variants, tTS and pTRE-Tight vector in transgenic mouse modeling
Though Tet-based systems have defect,
i.e. basal transgene leak derived from inherent defects in Tet-based systems in
vitro and in vivo, increasing evidences showed that a more tightly
controlled regulatory system can be readily achievable in vitro [32-39], ev vivo [37,38] and in
vivo [13,40-44] when the advanced versions
(e.g. rtTA2S-M2 and rtTA2S-S2) of rtTA [45],
tetracycline-controlled transcriptional silencer (tTS) [46] and an
"ideal" minimal promoter in responsive components (pTRE-Tight) (http://www.bdbiosciences.com/clontech/techinfo/vectors/vectorsT-Z/pTRE-Tight.shtml)
are employed alone or in combination therein. For instance, more stringent
control of transgene expression using improved versions of rtTA, for example
rtTA2S-S2
[40,41] and rtTA2S-M2 [17,42,43], has also been achieved in
transgenic mice for Cre recombinase [40], ferritin H [41], pigment
epithelium-derived factor (PEDF) [42] and lac Z [17,43]. tTS can efficiently
reduce or completely eliminates background expression in transgenic mice by
combining tTS with rtTA [13]. Information on the countermeasures available
to eliminate basal transgene leak of Tet-based systems has recently been summarized
[10]. We suppose that these properties would also allow the generation of
transgenic mice with pre-selected expression windows.
Combinatorial use of tTS
system and Tet-on advanced system in transgenic mouse modeling requires the
generation of another transgenic strain harboring the tTS transgene under the
control of a ubiquitous promoter or a cell-type/tissue-specific promoter. tTS
transgenic mice are crossed with bi-transgenic mice (e.g. rtTA-advanced/target
gene mice) to produce triple transgenic offspring, allowing more tight control
of transgene expression. Although an obvious disadvantage of this approach is
the need to generate triple transgenic animals, there is the alternative of
combining the two control elements (i.e. rtTA-advanced and tTS) in a single
transgene under the control of a ubiquitous promoter or a cell-type or
tissue-specific promoter to produce rtTA-IRES-tTS transgenic mice (Fig. 2).
This "two-in-one" and "all-in-one strategy have been confirmed in
vitro and ev vivo [32-34,37,38].
General strategies for more
stringent control of transgene expression by the improved Tet-on system in
transgenic mouse modeling are fully presented in Fig. 2. The strategy of
crossing effector & silencer transgenic mice and acceptor transgenic mice
is more convenient and flexible because researchers are entitled to make full
use of a huge database of the different ubiquitous or cell/tissue-specific rtTA
or rtTA-advanced-tTS lines (http://www.zmg.unimainz.de/tetmouse) to
examine the transgene effects. At present,�
investigators are required to produce the acceptor transgenic mice
harboring gene(s) of interest, and subsequently mate them with a selected
effector and silencer from the database to attain bi-transgenic lines. In a
word, the developed and developing cell-type/tissue-specific rtTA-advanced-tTS
transgenic lines can be combined with other transgenic responder lines for more
tight and cell-type/tissue-specific over-expression of any target gene(s).
Co-injection strategy��� Furthermore, using one transgene system in which the tTS
construct, rtTA-advanced construct and TREmod-transgene construct are all
co-microinjected or integrated in a single transgenic construct is less time
consuming, less labor consuming, and at the same time avoids the segregation of
control elements during breeding. When two or several transgenic constructs are
co-injected into single cell fertilized embryos, in general, the co-injected
constructs are typically co-integrated into the same site(s) of the genome
[47]; since transgenes often tend to insert into the genome in a head to tail
pattern, this would provide transgene (+) animals with tight transgene control
in which all constructs are passed to progeny as if they are a single gene. This
would minimize the breeding and genotyping required for phenotypic
analysis. For example, simultaneous microinjection of CC10-rtTA, CC10-tTS and
tetO-TGF-b1 constructs successfully generated an inducible TGF-b1 mouse model
in Elias's laboratory [11].
"All-in-one"
strategy��� Some investigators integrated the two
expression units of Tet-off system [48] or Tet-on system [49-51] on a single DNA fragment in
transgenic mouse modeling, and demonstrated that this "two-in-one"
system is functional in transgenic mice. In addition, a simple
"all-in-one" vector, containing all of the elements of the
Dox-inducible Tet-on system in their most advanced variants (rtTA2S-M2 and
tTS), can be used to control transgene expression efficiently in long-term
tissue culture and in the mouse hematopoietic system following bone marrow
transplantation [32]. Bornkamm et al. [33] and Epanchintsev et al.
[34] achieved more stringent Dox-dependent control of gene activities and
shRNAmir expression (see below for more details) in vitro using an
episomal one-vector system (pRTS-1, a simple "all-in-one" vector),
which carries all the elements (including rtTA2S-M2, tTS and the bidirectional
promoter P(tet)bi-1 in the same transgene construct), respectively. The vector
of pRTS-1 was confirmed to be low background activity in vitro [33].
However, whether this "all-in-one" system is functional in transgenic
mice remains to be fully elucidated. Anyway, in the first place, this
"two-in-one" or "all-in-one" strategy eliminates the need
to generate two independent transgenic lines and greatly facilitates the mouse
breeding strategy as effector and responder will not segregate; in addition,
integrating an equal number of target and transactivator genes may allow for a
more accurately quantitative regulation of purposed gene expression.
Temporal, spatial and lineage-specific gene
expression accomplished by integrating Tet-based systems
and Cre/lox P system
Two powerful systems,
tetracycline-inducible systems and Cre/lox P system, are extensively and
successfully employed in the transgenic mouse modeling; tetracycline-inducible
systems allow a reversible/temporal regulation of transgene expression by
addition of Dox to or withdrawal of Dox from drinking water, whereas Cre/lox
P system permits gene(s) to be permanently activated or inactivated in the
specific lineage at a specific time point [18,19]. Recently four individual
labs [14-17] have already developed a
unique and versatile transgenic mouse system based on (r)tTA dependent,
Dox-mediated regulation of target gene(s) following a Cre-deletion event to
realize temporal, spatial and lineage-specific gene expression in mice.
A triple, unique and versatile
system developed in the two individual labs [14,17] is well demonstrated in Fig.
3. In this system, for the reliable rtTA expression in a broad range of
cell types, rtTA-IRES-EGFP [14] or rtTA2S-M2 [17] transgene,
preceded by a lox P-flanked STOP sequence, is integrated into the ROSA26
(R26) locus to create a R26-STOP-rtTA-IRES-EGFP or R26-STOP-rtTA2S-M2
transgenic mouse strain with the rtTA or rtTA2S-M2 expression under the
regulation of an endogenous and ubiquitous R26 promoter, respectively.
Therefore, in this system, Cre-mediated deletion of STOP cassette from ROSA26
locus will immediately activate the rtTA [14] or rtTA2S-M2 [17] expression in the
Cre-expressing cells, and thereafter R26-rtTA gene activated by the excision of
STOP cassette remains active in all of their derivatives regardless of Cre
expression in these cells, in contrast to the Tet-based systems in which (r)tTA
is expressed directly under the control of a ubiquitous promoter or a cell type
or tissue-specific promoter (Fig. 2). Thus, the use of a proper Cre
transgenic mouse line (available at http://www.mshri.on.ca/nagy)
enables the cell type-, tissue-, or lineage-specific expression of rtTA, in
other words, the promoter specificity of Cre transgene in mice determines where
the Tet-based system becomes active. In the Cre-expressing cells and their
daughter cells, the gene(s) under investigation is/are silent in the absence of
Dox; while exposure to Dox, the active rtTA activates the expression of any
given gene(s). Collectively, after Cre-mediated deletion of STOP cassette, the
expression of target transgene is further controlled by Tet-on system in a
temporal and lineage-specific manner. For example, the ability of this
versatile system for targeted gene expression was primarily demonstrated in the
neuroepithelial and hematopoietic lineages, and in the derivatives of neural
crest and in the mammary epithelium [17] and in neural crest linage [14].
Moreover, other two labs
[15,16] developed another advanced and versatile system in which a Tet-off
regulation unit as a single cassette was readily integrated into the ROSA26
locus of mouse embryonic stem cells (ES cells) and becomes active after
Cre-mediated excision of STOP cassette in the Cre-expressing cells and their
descendants. In the system developed by Miyazaki's lab [16], the tTA expression
is conditional to a Cre-mediated excision of a STOP cassette (inserted between
promoter and tTA gene) from the ROSA26 locus [16], whereas the other developed
by McMahon's lab [15], a STOP cassette is inserted between PminCMV promoter and target gene(s).
Using the approach, the feasibility of dual regulatory drug control in the
regulation of targeted allele by manipulating Hh signaling with a SmoM2-YFP
allele was successfully tested in McMahon' lab [15]; in this lab, the
established Cre-lox P and Dox regulated 3-1 ES cell line is being used
to be spatio-temporal manipulation of each of five major development-related
signaling pathways, such as Hedgehog, Wnt, BMP, FGF and Notch, to aid in
genetically exploring the complex processes in vitro and in vivo [15].
The strategies and approaches
presented here abrogates the need to produce and characterize new transgenic
mouse lines expressing (r)tTA in the desired tissue or cell-type specific
pattern where a large number of well-characterized cell-type
or tissue-specific Cre transgenic mouse strains already exist in a voluntary
database of Cre-expressing mice (http://www.mshri.on.ca/nagy).
These unique and versatile systems will be useful for investigating genes, in
which time-dependent regulation of transgene expression is pretty necessary for
fully exploring the full spectrum of gene functions within descendants of a
specific lineage with a great flexibility, which is pretty difficult to achieve
with the conventional systems, such as Tet-based systems alone or Cre/lox
P system only.
Reversible, temporal and spatial gene silencing by shRNAmir expression in the transgenic mouse modeling
The advent of RNA interference
(RNAi) has led to the ability to revolutionize the functional analysis of genes
in vitro and in vivo. The vector-based RNAi has already been used
to successfully achieve the specific and stable knockdown of endogenous genes
with high efficiency in the transgenic mice [52]. A number of vector-based
Cre-controlled transgenic RNAi systems for conditional RNAi have been described
previously [53-58]. Temporal and/or spatial
knockdown of endogenous gene expression with high efficiency in mice can be
reproducibly realized by conditionally controlling of siRNA expression using
the Cre/lox P system [56-59]. Increasing data
demonstrated that stable and efficient knockdown of endogenous gene expression
in mice through vector-based transgenic RNAi strategy can generate phenotypes
of gene knockout in mice [59-61]. Together, the
vector-based transgenic RNAi strategy offers a technically simpler, cheaper and
quicker alternative to classical gene knockout technology in deciphering gene
functions in vivo in a temporal and/or spatial manner.
Although limited choices of
RNA polymerase (Pol) III promoters have been developed to express shRNA, a
large repertoire of Pol II promoters have been successfully used in transgenic
mice. microRNAs (miRNAs), the endogenous form of shRNAs that carry out gene
silencing function, are downstream of Pol II promoters and are expressed by Pol
II activity [61,62]. microRNA-adapted shRNA (shRNAmir) construct, containing
Pol II promoter driving the expression of an shRNA with a structure that mimics
human miRNA miR-30a, has been successfully designed to mimic a natural miRNA primary
transcript, enabling specific processing by the endogenous RNAi pathway and
producing more effective and specific gene knockdown derived from
microRNA-30 adapted design [61,62].
Recently, the expression
arrest shRNAmir libraries, which were designed to facilitate the
RNAi-mediated down-regulation of all human or mouse genes, were
developed [62]. A large collection of human and mouse pSM2-based shRNAmir
sequences are publicly available from Open Biosystems (http://www.openbiosystems.com), which
can be easily transferred from pSM2c vector to the vector of interest by the
ligation-free MAGIC technique [63].
Temporal, spatial and
lineage-specific RNAi-mediated gene silencing accomplished by integrating
Tet-based systems and Cre/lox P system
Recently, a series of
lentiviral vectors (called pPRIME, potent RNAi using shRNAmir expression),
which provide high penetrance Tet-regulatable knockdown of gene expression at
single copy in vitro, are newly developed in Prof. Elledge's lab [64]. If integrating this
pPRIME system with RNAi regulated by Tet-based systems and the versatile system
mentioned above [14,17], the temporal, spatial and lineage-specific loss of
gene function in mice can be readily accomplished (Fig. 4), which would
facilitate analyzing the full spectrum of gene functions within descendants of
a specific lineage with a huge flexibility, whereas the strategy for
Cre-lox-regulated conditional RNAi allows gene(s) of interest to be permanently
silenced or activated. Moreover, unlike gene knockout, RNAi strategy does not
destroy the gene structure.
Stringent and temporal
RNAi-mediated down-regulation of target gene by the improved Tet-based system
Epanchintsev et al. [34]
generated an all-in-one episomal vector (pEMI, adapted for convenient
ligation-free transfer of microRNA cassettes from public libraries) for
simultaneously conditional shRNAmir expression and a fluorescent marker protein
by modifying the pRTS-1 vector described above [33]. This conditional
knockdown-system of the pEMI vector has been confirmed to stringently,
reversibly and temporally regulate the expression of miRNAs mediating
RNAi, followed by down-regulation of target gene(s), thereby providing a convenient
tool to determine gene functions. One major advantage of the system described
here is the possibility to realize the conditional knock-down of the
gene/protein of interest in one step as all components have been integrated on
a single vector. pEMI vector is compatible with recently generated microRNA
public libraries (http://www.openbiosystems.com)
and will therefore presumably become a widely used tool for conditional RNAi.
Moreover, integrating pEMI
vector and Cre/lox P system by inserting a
lox P-flanked transcription STOP cassette sequence (SCS) [18,19] between the
bidirectional promoter [P(tet)bi-1] and miR30-shRNA will also permit a
temporal, spatial and lineage-specific loss of gene function by
shRNAmir-mediated RNAi.
Conclusion and prospects
The Tet regulatory systems are currently the most widely used regulatory systems for conditional gene expression. Ongoing improvements of the existing components and the continuous addition of new components or other systems, such as Cre/lox P system, to expand its range of applicability will make the Tet-based inducible systems more tight, versatile and flexible. Particular applications will include modeling the complex regulatory setups required to analyze sophisticated and multifactorial biological processes in development and disease, consequently not only improving our understanding of living organisms, but also demonstrating some novel and innovative strategies and approaches for the treatment of various human diseases.
References
1��� Gossen
M, Bujard H. Tight control of gene expression in mammalian cells by
tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992, 89: 5547-5551
2��� Gossen
M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. Transcriptional
activation by tetracyclines in mammalian cells. Science 1995, 268: 1766-1769
3��� Albanese
C, Hulit J, Sakamaki T, Pestell RG. Recent advances in inducible expression in
transgenic mice. Semin Cell Dev Biol 2002, 13: 129-141
4��� Bockamp
E, Maringer M, Spangenberg C, Fees S, Fraser S, Eshkind L, Oesch F et al.
Of mice and models: Improved animal models for biomedical research. Physiol
Genomics 2002, 11: 115-132
5��� Lewandoski
M. Conditional control of gene expression in the mouse. Nat Rev Genet 2001, 2:
743-755
6��� Ryding
AD, Sharp MG, Mullins JJ. Conditional transgenic technologies. J Endocrinol
2001, 171: 1-14
7��� van
der Weyden L, Adams DJ, Bradley A. Tools for targeted manipulation of the mouse
genome. Physiol Genomics 2002, 11: 133-164
8��� Baron
U, Bujard H. Tet repressor-based system for regulated gene expression in
eukaryotic cells: principles and
advances. Methods Enzymol 2000, 327: 401-421
9��� Berens
C, Hillen W. Gene regulation by tetracyclines. Constraints of resistance regulation
in bacteria shape TetR for application in eukaryotes. Eur J Biochem 2003, 270:
3109-3121
10�� Xiao D, Sun Y, Gu WW, Chen XG.
Tetracycline-controlled transcriptional regulation systems: Countermeasures to
eliminate basal transgene leak of Tet-based systems. Progress in Natural
Science 2007, 17: 11-19
11�� Zhu Z, Zheng T, Lee CG, Homer RJ, Elias JA.
Tetracycline-controlled transcriptional regulation systems: advances and
application in transgenic animal modeling. Semin Cell Dev Biol 2002, 13: 121-128
12�� Xiao D, Xu K, Yue Y, Guo ZM, Huang B, Deng
XY, Tang H et al. Temporal and tight hepatitis C virus gene activation
in cultured human hepatoma cells mediated by a cell-permeable Cre recombinase.
Acta Biochim Biophys Sin 2004, 36: 687-694
13�� Zhu Z, Ma B, Homer RJ, Zheng T, Elias JA. Use
of the tetracycline-controlled transcriptional silencer (tTS) to eliminate
transgene leak in inducible overexpression transgenic mice. J Biol Chem 2001,
276: 25222-25229
14�� Belteki G, Haigh J, Kabacs N, Haigh K, Sison
K, Costantini F, Whitsett J et al. Conditional and inducible transgene
expression in mice through the combinatorial use of Cre-mediated recombination
and tetracycline induction. Nucleic Acids Res 2005, 33: e51
15�� Mao J, Barrow J, McMahon J, Vaughan J,
McMahon AP. An ES cell system for rapid, spatial and temporal analysis of gene
function in vitro and in vivo. Nucleic Acids Res 2005, 33: e155
16�� Miyazaki S, Miyazaki T, Tashiro F, Yamato E,
Miyazaki J. Development of a single-cassette system for spatiotemporal gene regulation
in mice. Biochem Biophys Res Commun 2005, 338: 1083-1088
17�� Yu HM, Liu B, Chiu SY, Costantini F, Hsu W.
Development of a unique system for spatiotemporal and lineage-specific gene
expression in mice. Proc Natl Acad Sci USA 2005, 102: 8615-8620
18�� Nagy A. Cre recombinase: The universal
reagent for genome tailoring. Genesis 2000, 26: 99-109
19�� Sauer B. Inducible gene targeting in mice
using the Cre/lox system. Methods 1998, 14: 381-392
20�� Lee P, Morley G, Huang Q, Fischer A, Seiler
S, Horner JW, Factor S et al. Conditional lineage ablation to model
human diseases. Proc Natl Acad Sci USA 1998, 95: 11371-11376
21�� Wakita T, Taya C, Katsume A, Kato J, Yonekawa
H, Kanegae Y, Saito I et al. Efficient conditional transgene expression
in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP
system. J Biol Chem 1998, 273: 9001-9006
22�� Machida K, Tsukiyama-Kohara K, Seike E, Tone
S, Shibasaki F, Shimizu M, Takahashi H et al. Inhibition of cytochrome c
release in Fas-mediated signaling pathway in transgenic mice induced to express
hepatitis C viral proteins. J Biol Chem 2001, 276: 12140-12146
23�� Takaku S, Nakagawa Y, Shimizu M, Norose Y,
Maruyama I, Wakita T, Takanot et al. Induction of hepatic injury by
hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic
mice expressing the viral structural genes. Biochem Biophys Res Commun 2003,
301: 330-337
24�� Wakita T, Katsume A, Kato J, Taya C, Yonekawa
H, Kanegae Y, Saito I et al. Possible role of cytotoxic T cells in acute
liver injury in hepatitis C virus cDNA transgenic mice mediated by Cre/loxP
system. J Med Virol 2000, 62: 308-317
25�� Chisari FV, Pinkert CA, Milich DR. A
transgenic mouse model of the chronic hepatitis B surface antigen carrier
state. Science 1985, 230:1157-1160
26�� Tan WJ, Chen G, Li GS, Cong Y, Miao J, Du M,
Zhan MY. Production of three transgenic mouse lineages which simultaneously
carries structural gene and/or nonstructural gene3 (NS3) region of hepatitis C
virus. Prog Biochem Biophys 1998, 25: 279-282
27�� Tan WJ, Lang ZW, Cong Y, Yi Y, Zhan MY.
Transgenic expression of hepatitis C virus core and NS3 proteins in the mouse
is not cytopathic. Chinese Journal of Virology 1998, 14: 302-306
28�� Dandri M, Lutgehetmann M, Volz T, Petersen J.
Small animal model systems for studying hepatitis B virus replication and
pathogenesis. Seminars In Liver Disease 2006, 26: 181-191
29�� Milich DR. Transgenic technology and the
study of hepatitis viruses: a
review of what we have learned. Can J Gastroenterol 2000, 14: 781-787
30�� Akbar SK, Onji M. Hepatitis B virus
(HBV)-transgenic mice as an investigative tool to study immunopathology during
HBV infection. Int J Exp Pathol 1998, 79: 279-291
31�� Feitelson MA, Larkin JD. New animal models of
hepatitis B and C. ILAR J 2001, 42: 127-138
32�� Barde I, Zanta-Boussif MA, Paisant S, Leboeuf
M, Rameau P, Delenda C, Danos O. Efficient control of gene expression in the
hematopoietic system using a single Tet-on inducible lentiviral vector. Mol
Ther 2006, 13: 382-390
33�� Bornkamm GW, Berens C, Kuklik-Roos C, Bechet
JM, Laux G, Bachl J, Korndoerfer M et al. Stringent
doxycycline-dependent control of gene activities using an episomal one-vector
system. Nucleic Acids Res 2005, 33: e137
34�� Epanchintsev A, Jung P, Menssen A, Hermeking
H. Inducible microRNA expression by an all-in-one episomal vector system.
Nucleic Acids Res 2006, 34: e119
35�� Knott A, Garke K, Urlinger S, Guthmann J,
Muller Y, Thellmann M, Hillen W. Tetracycline-dependent gene regulation: combinations of transregulators yield a
variety of expression windows. Biotechniques. 2002, 32: 796, 798, 800
36�� Koponen JK, Kankkonen H, Kannasto J, Wirth T,
Hillen W, Bujard H, Yla-Herttuala S. Doxycycline-regulated lentiviral vector
system with a novel reverse transactivator rtTA2S-M2 shows a tight control of
gene expression in vitro and in vivo. Gene Ther 2003, 10: 459-466
37�� Mizuguchi H, Xu ZL, Sakurai F, Mayumi T,
Hayakawa T. Tight positive regulation of transgene expression by a single
adenovirus vector containing the rtTA and tTS expression cassettes in separate
genome regions. Hum Gene Ther 2003, 14: 1265-1277
38�� Salucci V, Scarito A, Aurisicchio L,
Lamartina S, Nicolaus G, Giampaoli S, Gonzalez-Paz O et al. Tight control
of gene expression by a helper-dependent adenovirus vector carrying the
rtTA2(s)-M2 tetracycline transactivator and repressor system. Gene Ther 2002,
9: 1415-1421
39�� Lena AM, Giannetti P, Sporeno E, Ciliberto G,
Savino R. Immune responses against tetracycline-dependent transactivators
affect long-term expression of mouse erythropoietin delivered by a
helper-dependent adenoviral vector. J Gene Med 2005, 7: 1086-1096
40�� Schonig K, Schwenk F, Rajewsky K, Bujard H.
Stringent doxycycline dependent control of CRE recombinase in vivo.
Nucleic Acids Res 2002, 30: e134
41�� Wilkinson J 4th, Di X, Schonig K, Buss JL,
Kock ND, Cline JM, Saunders TL et al. Tissue-specific expression of
ferritin H regulates cellular iron homoeostasis in vivo. Biochem J 2006,
395: 501-507
42�� Kerrison JB, Duh EJ, Yu Y, Otteson DC, Zack
DJ. A system for inducible gene expression in retinal ganglion cells. Invest
Ophthalmol Vis Sci 2005, 46: 2932-2939
43�� Michalon A, Koshibu K, Baumgartel K, Spirig
DH, Mansuy IM. Inducible and neuron-specific gene expression in the adult mouse
brain with the rtTA2S-M2 system. Genesis
2005, 43: 205-212
44�� Mallo M, Kanzler B, Ohnemus S. Reversible
gene inactivation in the mouse. Genomics 2003, 81: 356-360
45�� Urlinger S, Baron U, Thellmann M, Hasan MT,
Bujard H, Hillen W. Exploring the sequence space for tetracycline-dependent
transcriptional activators: Novel mutations yield expanded range and
sensitivity. Proc Natl Acad Sci USA 2000, 97: 7963-7968
46�� Freundlieb S, Schirra-Muller C, Bujard H. A
tetracycline controlled activation/repression system with increased potential
for gene transfer into mammalian cells. J Gene Med 1999, 1: 4-12
47�� Overbeek PA. Factors affecting transgenic
animal production. In: Pinkert CA ed. Transgenic Animal Technology: A
Laboratory Handbook. San Diego: Academic Press Inc 1994
48�� Schultze N, Burki Y, Lang Y, Certa U,
Bluethmann H. Efficient control of gene expression by single step integration
of the tetracycline system in transgenic mice. Nat Biotechnol 1996, 14: 499-503
49�� Juhila J, Roozendaal R, Lassila M, Verbeek
SJ, Holthofer H. Podocyte cell-specific expression of doxycycline inducible Cre
recombinase in mice. J Am Soc Nephrol 2006, 17: 648-654
50�� Lottmann H, Vanselow J, Hessabi B, Walther R.
The Tet-On system in transgenic mice: Inhibition of the mouse pdx-1 gene
activity by antisense RNA expression in pancreatic beta-cells. J Mol Med 2001,
79: 321-328
51�� Utomo AR, Nikitin AY, Lee WH. Temporal,
spatial, and cell type-specific control of Cre-mediated DNA recombination in
transgenic mice. Nat Biotechnol 1999, 17: 1091-1096
52�� Coumoul X, Deng CX. RNAi in mice: A promising
approach to decipher gene functions in vivo. Biochimie 2006, 88: 637-643
53�� Coumoul X, Li W, Wang RH, Deng C. Inducible
suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA
interference (RNAi) and the Cre-LoxP system. Nucleic Acids Res 2004, 32: e85
54�� Fritsch L, Martinez LA, Sekhri R, Naguibneva
I, Gerard M, Vandromme M, Schaeffer L et al. Conditional gene knock-down
by CRE-dependent short interfering RNAs. EMBO Rep 2004, 5: 178-182
55�� Kasim V, Miyagishi M, Taira K. Control of
siRNA expression using the Cre-loxP recombination system. Nucleic Acids
Res 2004, 32: e66
56�� Tiscornia G, Tergaonkar V, Galimi F, Verma
IM. CRE recombinase-inducible RNA interference mediated by lentiviral vectors.
Proc Natl Acad Sci USA 2004, 101: 7347-7351
57�� Ventura A, Meissner A, Dillon CP, McManus M,
Sharp PA, Van Parijs L, Jaenisch R et al. Cre-lox-regulated
conditional RNA interference from transgenes. Proc Natl Acad Sci USA 2004, 101:
10380-10385
58�� Yu J, McMahon AP. Reproducible and inducible
knockdown of gene expression in mice. Genesis 2006, 44: 252-261
59�� Coumoul X, Shukla V, Li C, Wang RH, Deng CX.
Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference.
Nucleic Acids Res 2005, 33: e102
60�� De Souza AT, Dai X, Spencer AG, Reppen T,
Menzie A, Roesch PL, He Y et al. Transcriptional and phenotypic
comparisons of Ppara knockout and siRNA knockdown mice. Nucleic Acids Res 2006,
34: 4486-4494
61�� Xia XG, Zhou H, Samper E, Melov S, Xu Z. Pol
II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of
the gene knockout in mice. PLoS Genet 2006, 2: e10
62�� Silva JM, Li MZ, Chang K, Ge W, Golding MC,
Rickles RJ, Siolas D et al. Second-generation shRNA libraries covering
the mouse and human genomes. Nat Genet 2005, 37: 1281-1288
63�� Li MZ, Elledge SJ. MAGIC, an in vivo
genetic method for the rapid construction of recombinant DNA molecules. Nat
Genet 2005, 37: 311-319
64�� Stegmeier F, Hu G, Rickles RJ, Hannon GJ,
Elledge SJ. A lentiviral microRNA-based system for single-copy polymerase
II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 2005,
102: 13212-13217