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J Biol Chem, Vol. 274, Issue 53, 38071-38082, December 31, 1999
From the Department of Molecular Biology and Pharmacology,
Washington University School of Medicine,
St. Louis, Missouri 63110
We have developed two systems for performing
Cre-mediated recombination of target genes in the rapidly self-renewing
mouse small intestinal and colonic epithelium. When expression of Cre recombinase is placed directly under the control of transcriptional regulatory elements from a fatty acid-binding protein gene
(Fabp), deletion of loxP flanked (floxed) DNA sequences is
initiated as early as embryonic day 13.5, well before completion of
intestinal morphogenesis. By embryonic day 16.5, Fabp-Cre
also directs recombination in all cell layers of the transitional
epithelium that lines the renal calyces and pelvis, ureters, and
bladder. Fabp-Cre expression and recombination are
maintained in both epithelia throughout adulthood. The second system
allows recombination to be induced only in the gut and at any period
during adulthood. This system uses Fabp regulatory elements
to direct expression of a reverse tetracycline-regulated transactivator
(rtTA). Another transgene encodes Cre under the control of
tet operator sequences and a minimal promoter from human
cytomegalovirus (tetO-PhCMV-Cre). In the
absence of a doxycycline inducer, no basal recombination is detectable
in the gut of adult tri-transgenic mice containing Fabp-rtTA, tetO-PhCMV-Cre, plus a
floxed reporter gene. After 4 days of oral administration of
doxycycline, recombination of the reporter is apparent in the small
intestinal, cecal, and colonic epithelium. After doxycycline is
withdrawn, the recombined locus persists for at least 60 days,
indicating that recombination has occurred in epithelial cell
progenitors that have long residency times in the proliferative units
of the intestine (crypts of Lieberkühn). This inducible system
should have a number of applications for examining gene function at
selected times in postnatal life, under selected physiologic or
pathophysiologic conditions.
The adult mouse intestinal epithelium undergoes continuous and
rapid renewal (1-6). It provides an attractive model system for
examining how proliferation, differentiation, and death programs are
coordinated to maintain several cell lineages. One way of auditing the
contribution of specific genes to these programs is by generating null
alleles using Cre recombinase expressed under the control of
lineage-specific transcriptional regulatory elements. This
site-specific integrase from bacteriophage P1 catalyzes recombination
at 34-bp1 loxP sites that
flank a gene segment to be deleted (7). A large toolbox of
transcriptional regulatory elements has been assembled that function in
the intestine, so it is now possible to express foreign gene products,
such as Cre, in specified regions of the cephalocaudal axis of the gut,
in selected cell lineages at various points in their differentiation
programs, and at various stages of development (e.g. Refs.
8-12). Even with these reagents in hand, the rapid and perpetual
renewal of this epithelium could pose a problem for those who wish to
perform efficient and persistent conditional gene knockouts.
Epithelial cell renewal in the adult mouse intestine is fueled by
multipotent stem cells located at, or near, the base of flask-shaped
mucosal invaginations known as crypts of Lieberkühn (5, 13, 14).
Crypts are dynamic structures. Their average lifespan is ~110
days (15, 16). Maintenance of crypts is dependent upon their stem cell
population. Once this population reaches a critical threshold and the
volume of a crypt exceeds a critical size, the crypt divides (17).
Division occurs by branching morphogenesis with asymmetric partitioning
of epithelial progenitors among the two daughter crypts (15, 17, 18).
The crypt stem cell has yet to be identified; one candidate is the
crypt base columnar cell described by Cheng and Leblond (5) during
lineage tracing studies that employed tritiated thymidine labeling
followed by EM autoradiography.
The committed daughters of the stem cells undergo several rounds of
division, forming a rapidly cycling transit cell population located in
the mid-portion of each adult crypt. The stem cell ultimately gives
rise to four cell types in the small intestine: enterocytes,
representing >80% of all epithelial cells, goblet cells,
enteroendocrine cells, and Paneth cells. The first three lineages
complete their terminal differentiation during a rapid (2-5-day) and
orderly migration from the crypt up an adjacent villus (1, 3, 4, 19).
As mature epithelial cells approach the villus tip, they are removed by
apoptosis or exfoliation. The Paneth cell lineage is the exception; it
differentiates during a downward migration to the crypt base (2, 13).
Paneth cells have a lifespan of ~20 days (2) and are absent from
colonic crypts. The colon also lacks villi; differentiated epithelial cells emerge from a colonic crypt and form a flat hexagonal-shaped surface epithelial cuff that surrounds the crypt orifice (19).
The magnitude of epithelial cell renewal in the intestine is
remarkable. On average, each small intestinal crypt produces ~300 new
cells per day, a number equivalent to its steady state population. The
~7000 epithelial cells that cover a villus located in the mid-portion
of the intestine are replaced every ~30-40 h (e.g. Ref.
6).
These features raise several questions for those contemplating using
Cre recombinase to orchestrate gene knockouts in the intestinal
epithelium, as opposed to cell populations that are non-renewing and/or
are long-lived. If Cre is expressed under the control of
transcriptional regulatory elements that only operate in
differentiating intestinal epithelial cells, will the speed of recombination be rapid enough so that the gene of interest will be
silenced and its product lost before these cells are removed? How many
active stem cells does a given crypt contain? Do they have equivalent
mitotic rates? Do they have similar or dissimilar residence times in
the crypt? If Cre is not directed to all active or potential crypt stem
cells, how likely is it that within its lifespan a crypt will become
populated by a mixture of cells, some with and some without the
Cre-engineered recombined (null) allele?
In this report, we establish the feasibility of performing efficient
Cre-mediated gene knockouts in colonic crypts. A system for inducing
gene knockouts in the colonic epithelium at any time during postnatal
life is described. This latter system has allowed us to explore whether
a recombined null allele can persist for more than one cycle of
epithelial renewal and, coincidentally, to examine the residence time
of epithelial progenitors within crypts.
Generation of Transgenic Mice
Fabpl4× at Fabpl4× at
Fabpl4× at tetO-PhCMV-Cre--
This construct contains 7 copies
of the tet operator sequence (7×tetO).
7×tetO is located upstream of a minimal promoter from human
cytomegalovirus IEI (PhCMV; Ref. 23), which is
followed by (i) the Cre recombinase ORF surrounded by 48-bp FRT sites
recognized by FLP recombinase (24) and (ii) an intron and
polyadenylation sequence from the human ENHmov (loxP)hygroR(loxP)--
Mice
homozygous for ENHmov (25) were a gift from Shirley
Tilghman (Princeton University). They were produced from ES cells transfected with a targeting vector that allowed a set of two enhancers, normally located 3' to the H19 gene, to be moved
to a position equidistant between H19 and the closely linked
Igf2 gene on chromosome 7. A floxed hygromycin
resistance gene ((loxP)hygroR(loxP)) was
included next to the displaced enhancers when the ENHmov
allele was engineered.
Maintenance of Transgenic Mice--
All mice used in this study
were housed in microisolator cages under a strict light cycle (lights
on at 0600 h and off at 1800 h). Mice were given a standard
irradiated chow diet (PicoLab Rodent Chow 20, Purina Mills Inc.)
ad libitum. Animals were maintained in a specified
pathogen-free state. All FVB/N pedigrees were hemizygous for their
transgenes. Members of each pedigree were identified by PCR. hGH DNA
sequences were detected using 5'-AGGTGGCCTTTGACACCTACCAGG-3' and
5'-TCTGTTGTGTTTCCTCCCTGTTGG-3' as primers. This primer pair, which
spans intron 2 of the hGH gene, produces a 360-bp product. Cre DNA
sequences were identified with 5'-CCGGTTATTCAACTTGCACC-3' and
5'-CTGCATTACCGGTCGATGCAAC-3' which generate a 149-bp PCR product. rtTA
DNA sequences were detected with 5'-CGCCCAGAAGCTAGGTGTAG-3' and
5'-GCTCCATCGCGATGACTTAG-3' which produce a 200-bp product. Cycle
conditions were as follows: denaturation, 94 °C for 1 min; annealing, 55 °C for 1 min; extension, 72 °C for 1.5 min, for a
total of 30 cycles.
RNase Protection Assays--
cRNA probes for detecting mRNA
transcripts containing Cre or rtTA sequences were prepared as follows.
A 400-bp BamHI/ClaI fragment from the Cre
recombinase gene was subcloned into
BamHI/ClaI-digested pBluescript SK+. A 232-bp
StyI/SalI fragment from rtTA was subcloned into
SalI/XbaI-digested pBluescript SK+. In
vitro transcriptions were performed with XhoI-digested
plasmid DNA as the template, T7 RNA polymerase, and
[32P]CTP (NEN Life Science Products), together with
reagents and protocols supplied in the MAXIscript kit (Ambion, Inc).
Labeled cRNA was hybridized overnight at 50 °C to 15 µg of a total
cellular tissue RNA preparation. The products were then added to
reaction mixtures containing Tris-HCl (pH 7.5, final concentration 10 mM), NaCl (300 mM), EDTA (5 mM),
RNase A (40 µg/ml; 100 units/mg; Sigma) and RNase T1 (2 µg/ml;
10,000 units/mg; Sigma). Following a 1-h incubation at 37 °C, RNA
was extracted, precipitated with ethanol, and subjected to
electrophoresis through urea/acrylamide gels.
A positive control was included in all RNase protection assays. A
270-bp XhoII/DraI fragment of the mouse
rpl32 gene was subcloned into pBluescript SK+. The resulting
recombinant plasmid was linearized with BamHI, and
rpl32 cRNA was synthesized using T3 RNA polymerase.
Isolation of RNA from the Intestinal Epithelium and
Mesenchyme--
The proximal and distal small intestine, cecum, and
colon were opened along their cephalocaudal axes, and their luminal
contents were removed by washing the tissues in phosphate-buffered
saline (PBS). Each segment was placed in Hanks' buffered saline
solution (HBSS) containing 25 mM HEPES and 1% calf serum
(Sigma) (3 washes of 5 min each, 20 ml/wash). The washed segments were
put in sterile polypropylene tubes containing 20 ml of HBBS/50
mM EDTA, and the tubes were rotated at 100 rpm for 15 min.
Tissue fragments were allowed to settle, and the supernatant was
removed and saved on ice. The settled tissue fragments were subjected
to two more cycles of agitation in HBBS/EDTA, followed by settlement.
Supernatants were pooled and spun at 250 × g for 10 min at 4 °C. RNA was isolated (RNeasy kit, Qiagen) from the cell
pellets (epithelial fraction) and the remaining tissue fragments
(mesenchymal fraction) (26). Some fragments from each mesenchymal
preparation were fixed in periodate/lysine/paraformaldehyde (PLP)
embedded in paraffin, and 5 µm-thick sections were cut. These
sections were stained with hematoxylin and eosin and examined to verify
that all the epithelium had been removed.
Assays for Recombination
PCR--
A forward primer (HyF, 5'-GAGTAGAGGCTTGATCAGGGC-3') and
two reverse primers (HyR1, 5'-CCTCACAGCACTACCCTGAGAG-3' and HyR2, 5'-CTTCCATTGCTCAGCGGTG-3') were used to assay for recombination at the
floxed hygroR locus. HyF and HyR2 amplify a
680-bp fragment from the intact (unrecombined) locus. HyF and HyR1
amplify a 475-bp fragment from the recombined locus. PCR reactions
contained 100-250 ng of DNA.
Three primers were used to detect recombination of the
Fabpl4× at
Histochemical and Immunohistochemical Assays of Cre-mediated
Recombination of Fabpl4× at
Tissue sections were also stained with X-Gal. 5-8 µm-thick sections,
cut from frozen blocks of PLP-fixed tissues, were thawed at room
temperature for 20 min, washed in PBS, incubated overnight at 30 °C
in X-Gal solution, and then counter-stained with nuclear fast red (Sigma).
To detect hGH synthesized after recombination, using light microscopy,
the ileum, cecum, colon, and bladder were fixed in PLP for 45 min,
embedded in paraffin, and 5 µm-thick sections were prepared. These
sections were de-paraffinized with xylene, rehydrated, and pretreated
for 15 min at room temperature with PBS-blocking buffer (bovine serum
albumin (1%), Triton X-100 (0.3%), and CaCl2 (1 mM) in PBS). Sections were incubated subsequently with
rabbit anti-hGH (Dako; diluted 1:200 in PBS-blocking buffer for
intestinal sections; 1:5000 for bladder sections). Antigen-antibody complexes were visualized with indocarbocyanine (Cy3)-conjugated donkey
anti-rabbit Ig (Jackson ImmunoResearch; final dilution in PBS-blocking
buffer = 1:500). Nuclei were stained with bisbenzimide (50 ng/ml
PBS).
To detect hGH by EM, mice were anesthetized, and a solution of PBS
containing paraformaldehyde (2%) and glutaraldehyde (2%) was infused
into their left ventricle. The bladder was post-fixed with the
perfusion buffer, dehydrated with graded ethanols, and embedded in
Lowicryl (Electron Microscopy Sciences). 500-nm thick sections were
cut, placed on glass slides, and counterstained with toluidine blue
(Electron Microscopy Sciences) for light microscopy. Adjacent 50-80
nm-thick sections were placed on slotted Formvar-coated grids (Electron
Microscopy Sciences), and the grids were floated for 30 min at room
temperature on a solution of Tris-buffered saline/blocking buffer (Tris
(20 mM, pH 7.4), NaCl (150 mM), normal mouse
serum (10%; Jackson ImmunoResearch), and Tween 20 (0.3%)). The grids
were then incubated for 2 h at room temperature with rabbit
anti-hGH (Dako; diluted 1:100 in Tris-buffered saline-blocking buffer).
Following washes with Tris-buffered saline, 0.3% Tween 20, antigen-antibody complexes were visualized with goat anti-rabbit IgG
containing bound 18-nm colloidal gold particles (Jackson
ImmunoResearch; final dilution = 1:15). Grids were counterstained
with aqueous uranyl acetate and lead and viewed with a JOEL model X100
electron microscope.
Inducible Gut-specific Knockouts
Tri-transgenic mice were generated by crossing male FVB/N mice
containing Fabpl4× at Generation of Transgenic Mice That Express Cre Recombinase in
Their Distal Small Intestine, Cecum, and Colon
We wanted to target gene knockouts to the distal small intestinal
and colonic epithelium since these regions of the gut are the sites of
a number of human diseases, including inflammatory bowel disease and
cancer. Therefore, we chose to express Cre recombinase under the
control of transcriptional regulatory elements derived from a fatty
acid-binding protein gene. These elements consist of nucleotides DNA encoding Cre recombinase with a nuclear localization signal
was placed between Fabpl4× at RNase protection assays of intestinal RNAs prepared from 6-8-week-old
members of six Fabpl4× at Cre-mediated Recombination Is Limited to the Distal Small
Intestine, Cecum, Colon, Ureters, and Bladder of Adult Bi-transgenic
Mice
FVB/N Fabpl4× at
Inducible Gene Knockouts in the Small Intestinal and Colonic
Epithelium*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
132/Cre--
This recombinant DNA
consists of the following: (i) nucleotides 
596 to +21 of the rat
Fabpl gene, with four additional tandem repeats of its
nucleotides 172 to 133 added at nucleotide 132 (Ref. 11;
abbreviated Fabpl4× at 132) linked to (ii) a
1.0-kb fragment, containing the Cre recombinase gene with a nuclear
localization signal from SV40 large T antigen (Ref. 20; a gift from
Gail Martin, University of California, San Francisco), and (iii)
nucleotides +3 to +2150 of the human growth hormone gene (hGH). Six
pedigrees of FVB/N Fabpl4× at 132/Cre mice
were established.
132/(loxP)lacZ(loxP)-hGH--
pFabpl4XlacZhGH is a
recombinant pBluescript SK+ plasmid that was generated through a
multistep process. It contains an insert consisting of
Fabpl4× at 132 linked to a 34-bp loxP
site, which in turn is linked to the Escherichia coli
-galactosidase gene (lacZ). The lacZ sequence
is followed by another loxP site and then nucleotides +3 to +2150 of
hGH. The Fabpl4× at
132/(loxP)lacZ(loxP)-hGH insert in pFabpl4XlacZhGH
was removed as a 6.8-kb NotI/SalI fragment and
injected into FVB/N oocytes. Four pedigrees of mice were established
with this transgene.
132/rtTA--
pUHG15-1-rTA (a gift
from Michael Rosenberg, Glaxo Wellcome) contains a mutant
tetracycline-controlled transactivator (rtTA) (21). The rtTA open
reading frame (ORF) was excised from pUHG15-1-rTA as a 1.0-kb
XbaI/BamHI fragment. (Note that XbaI
cleaves just after the initiator ATG codon.) The nucleotide sequence
upstream of the ORF was designed according to rules defined by Kozak
(22), in an attempt to optimize rtTA mRNA translation. To create
this sequence, two oligodeoxynucleotides (5'-GATCCACCATGT-3' and
5'-CTAGACATGGTG-3') were annealed to one another, and the resulting
double-stranded linker was ligated to (i) the rtTA
XbaI/BamHI fragment and (ii) pJS1 (pBluescript
SK+-Fabpl4× at 132/hGH) which had been
linearized by BamHI cleavage between its Fabpl4× at 132 and hGH elements. The
Fabpl4× at 132/rtTA/hGH insert contained in
the resulting plasmid, pColon-rtTA, was excised as a 4.0-kb
EcoRI fragment and used for injections into FVB/N oocytes.
Two pedigrees of FVB/N Fabpl4× at 132/rtTA
mice were characterized.
-actin gene. Seven pedigrees
of FVB/N transgenic mice were established containing this recombinant DNA.
132/(loxP)lacZ(loxP)-hGH transgene:
Fabpl forward primer (fLF;
5'-CTAGAGATGTGATTCACATG-3'); lacZ reverse primer (lacZR,
5'-CTTCGCTATTACGCCAGCTG-3'); and hGH reverse primer (hGHR,
5'-CAAACTCCTGGTAGGTGTCAAAG-3'). fLF and lacZR amplify a 375-bp fragment
from the intact transgene, and fLF and hGHR amplify a 302-bp fragment
when lacZ has been removed by Cre-mediated recombination.
The same thermocycling conditions were employed for all PCR assays of
recombination (denaturation, 94 °C for 1 min; annealing, 55 °C
for 1 min; extension, 72 °C for 1.5 min, for a total of 30 cycles).
132/(loxP)lacZ(loxP)-hGH--
E. coli
-galactosidase was detected by 5-bromo-4-chloro-3-indolyl
-D-galactoside (X-Gal) staining. The small intestine, cecum, colon, and bladder were flushed with PBS, cut open along their
cephalocaudal axes, and pinned on wax slabs. These wholemount preparations were fixed for 45 min at room temperature in PLP, washed
three times in PBS, and incubated in a solution containing dithiothreitol (20 mM), Tris (150 mM, pH 8.0),
and ethanol (20%) for 45 min at room temperature. Following three more
washes in PBS (5 min/wash), the wholemounts were incubated in X-Gal
solution overnight at 4 °C (X-Gal (2 mM; Roche Molecular
Biochemicals), potassium ferricyanide (4 mM), potassium
ferrocyanide (4 mM), and MgCl2 (2 mM); prepared in PBS, final pH of 7.6).
132/rtTA and either
(loxP)hygroR(loxP) or Fabpl4×
at 132/(loxP)lacZ(loxP)hGH with female mice
containing tetO-PhCMV-Cre. Recombination was
induced by adding doxycycline (Sigma, final concentration = 2 mg/ml) and sucrose (5% w/v) to their drinking water. A control group
of aged-matched (4-12-week-old) tri-transgenic mice received 5%
sucrose alone. All mice were treated for 4 days with drug or vehicle
alone. Animals were then sacrificed or they were maintained for an
additional 10, 20, 30, or 60 days on water that did not contain any
supplements. Recombination was surveyed in tissues using the PCR and
immunohistochemical assays described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
596
to +21 of rat Fabpl with 4 additional copies of a 35-bp
sequence, spanning nucleotides 177 to 133, that had been inserted
at nucleotide 132 (abbreviated Fabpl4× at
132) (11). Earlier light and EM immunohistochemical studies of several pedigrees of FVB/N transgenic mice had demonstrated that Fabpl4× at 132 can direct expression of a
human growth hormone (hGH) reporter throughout the epithelium of crypts
in the distal small intestine (ileum), cecum, and colon of adult mice
(11).
132 and
nucleotides +3 to +2150 of the hGH gene (encompassing all of its
exons and introns). Two in-frame stop codons were positioned between
the end of the Cre ORF and the initiator Met codon of hGH to prevent
synthesis of hGH from the mRNA product of Fabpl4×
at 132/Cre.
132/Cre transgenic
pedigrees revealed that 4 lines expressed Cre mRNA. Two pedigrees
were selected for further study. Six- to 10-week-old mice from both
pedigrees had indistinguishable patterns of transgene expression.
Steady state levels of Cre mRNA increased from the proximal to
distal thirds of their small intestine and were sustained in their
cecum and colon (n = 3 mice surveyed/pedigree; see Fig. 1). Expression was also prominent in
bladder and ureter but was not detectable in the renal parenchyma or in
11 other tissues (stomach, pancreas, liver, spleen, ovary or testes,
muscle, heart, lung, thymus, and brain) (Fig. 1 and data not shown).
This tissue-specific pattern of expression was maintained in
10-month-old animals (n = 2/pedigree).
View larger version (37K):
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Fig. 1.
Cre expression is limited to the
distal small intestine, cecum, colon, and bladder of adult
Fabpl4× at 132/Cre
transgenic mice. Total cellular RNA was isolated from
tissues harvested from a 10-week-old FVB/N transgenic mouse. The small
intestine was divided into proximal, middle, and distal thirds,
arbitrarily named duodenum, jejunum, and ileum. RNase protection assays
were performed using two labeled cRNAs: one that protects a
454-nucleotide domain from the Cre-containing mRNA transcript,
and another that protects a 270-nucleotide long segment of the
endogenous mouse rpl32 gene transcript.
132/Cre transgenic mice
were crossed to mice with a mixed C57Bl/6-129/Sv genetic background
containing a floxed hygromycin resistance gene
((loxP)hygroR(loxP)). Recombination was
monitored in bi-transgenic animals using a simple PCR assay (Fig.
2A). At 6-10 weeks of age,
recombination was evident in total cellular DNA isolated from jejunum,
ileum, cecum, colon, ureter, and bladder and was undetectable in DNA prepared from the other tissues listed above (Fig. 2B and
data not shown) (n = 6 mice). The same
tissue-specific pattern of recombination was documented in
10-month-old bi-transgenic mice. Control PCR assays of DNAs prepared
from animals that contained (loxP)hygroR(loxP)
without Fabpl4× at 132/Cre failed to
disclose recombination in any tissue, including the gut (data not
shown).
View larger version (53K):
[in a new window]
Fig. 2.
Tissue-specific recombination
of a floxed hygroR locus in adult
bi-transgenic mice containing
Fabpl4× at 132/Cre.
A, PCR primers used to monitor recombination at
hygroR. B, results of PCR assays of
total cellular DNAs prepared from the tissues of an 8-week-old
bi-transgenic mouse. The 475-bp fragment is derived from the recombined
hygroR locus, whereas the 680-bp fragment
represents the intact locus. Note that the distance between HyF and
HyR1 in the unrecombined allele is >2 kb.
PCR surveys of DNAs isolated from embryonic day 13.5 (E13.5), E14.5, E15.5, E16.5, and postnatal day 1 (P1), P7, and P14 bi-transgenic mice (n = 2-3/time point) revealed that recombination in the intestine commences as early as E13.5 (data not shown). Recombination in the bladder occurred as by E16.5 (the earliest time point surveyed).
Defining Cellular Patterns of Cre-mediated Recombination
Characterization of a Pedigree of Transgenic Mice That Is Useful
for Monitoring Cre-mediated Recombination in the Cecal, Colonic, and
Bladder Epithelium--
To examine the cellular basis of recombination
in the adult intestine (and bladder), we generated transgenic mice
containing the DNA construct shown in Fig.
3A. Fabpl4×
at 132 was placed upstream of an ORF encoding E. coli
lacZ with an added nuclear localization signal. The
lacZ gene was surrounded by loxP sites. Nucleotides +3 to
+2150 of the human growth hormone gene (hGH) were positioned downstream
of the lacZ ORF. The lacZ and hGH ORFs were
separated by three in-frame stop codons. There were several reasons why
we chose to use this transgene as a reporter of Cre-mediated
recombination. First, at the time of our study, there were no Cre
reporters that had been shown to function in the adult intestine and
bladder. Second, this reporter has the advantage of identifying
recombination in crypts by two criteria, loss of one foreign gene
product (E. coli -galactosidase) and gain of another
(hGH). Finally, expression of these two products is controlled by the
same Fabpl4× at 132 elements as those used to
express Cre. We reasoned that if Fabpl4× at
132/Cre transcription is initiated at a similar cell stratum
as Fabpl4× at
132/(loxP)lacZ(loxP)-hGH), then the relative
distributions of lacZ and hGH along the crypt will provide some measure
of the speed of Cre-mediated recombination. (By pulse labeling dividing
cells located in cecal and proximal colonic crypts with
5'-bromo-2-deoxyuridine, and then following their upward migration by
immunohistochemical analysis of sections prepared 1 h and 1, 2, 3, 5, and 7 days later, we had determined that it takes an average of only
5 days for cells to migrate to the surface epithelial cuff and to be
shed into the lumen (data not shown).
|
Four pedigrees of Fabpl4× at
132/(loxP)lacZ(loxP)-hGH transgenic mice were
generated. RNase protection assays of multiple tissue RNA samples
prepared from 6- to 8-week-old adult animals indicated that the
transgene was only expressed in one pedigree. Although lacZ-hGH
mRNA was present in the intestine of these mice, it was limited to
the cecum and colon; i.e. it was not detectable in distal
small intestinal RNA.2
Southern blot analysis indicated that the transgene had inserted at a
unique site in their genome and that there were ~2 copies of
Fabpl4× at
132/(loxP)lacZ(loxP)-hGH at this site.
Wholemount preparations of the cecum, colon, kidney, ureter, and
bladder from 6- to 24-week-old Fabpl4× at
132/(loxP)lacZ(loxP)-hGH transgenic mice
(n = 15) were stained with X-Gal to detect E. coli -galactosidase. X-Gal stained scattered patches of cecal
and colonic crypts (Fig. 4, A
and C). There was no detectable X-Gal staining of the cecal
or colonic epithelium of age-matched non-transgenic littermates (data
not shown), confirming that this staining was due to the product of
lacZ.
|
Surveys of serial sections of cecal and colonic crypts present in
6-12-week-old Fabpl4× at
132/(loxP)lacZ(loxP)-hGH mice (n = 4) revealed lacZ in epithelial cells positioned in the upper half of
crypts and in their associated surface epithelial cuffs (Fig.
4E). Sensitive immunohistochemical detection methods
did not reveal any hGH in the epithelium or mesenchyme (Fig.
4G).
E. coli -galactosidase was also apparent in the
transitional epithelium (urothelium) lining the renal calyces and
pelvis and ureters and bladder of Fabpl4× at
132/(loxP)lacZ(loxP)-hGH mice (Fig.
5, A and C).
Histochemical and immunohistochemical stains of sections prepared from
the bladder disclosed lacZ throughout the urothelium (Fig.
5E) and verified that hGH was absent (Fig. 5G).
X-Gal failed to stain the urothelium of age-matched nontransgenic
littermates (data not shown).
|
Cre-mediated Recombination in the Cecal and Colonic Crypt
Epithelium of Bi-transgenic mice--
Mice with
Fabpl4× at 132/Cre were crossed to mice with
Fabpl4× at
132/(loxP)lacZ(loxP)-hGH. Recombination in the
resulting bi-transgenic animals was assayed initially by PCR. The
tissue-specific pattern of recombination mirrored the tissue-specific
pattern of Cre expression, i.e. at 6-12 weeks of age,
recombination of the transgene was evident in jejunum, ileum, cecum,
colon, ureter, and bladder but was undetectable in stomach, renal
parenchyma without urothelium, skin, muscle, brain, thymus, lung,
heart, spleen, liver, pancreas, testes, or ovaries (e.g.
Fig. 3). PCR assays of control, age-matched mice containing
Fabpl4× at
132/(loxP)lacZ(loxP)-hGH alone showed no detectable
recombination in any of their tissues (data not shown).
These results were confirmed by histochemical staining. Examination of wholemount preparations of cecum and colon revealed that LacZ expression was largely eliminated in bi-transgenic mice, although some patches of positive (blue) crypts remained (Fig. 4, B, D, and F). Immunohistochemical studies of cecal and colonic sections indicated that loss of lacZ was accompanied by the appearance of hGH throughout the upper half of crypts and their associated surface epithelial cuffs (Fig. 4H).
The efficiency of recombination was evaluated from two perspectives:
the total number of crypts that were affected, and the extent of
recombination within a given crypt. To address the first issue, we
scored the number of X-Gal-positive and -negative crypts in cecal
wholemounts prepared from 6-week-old Fabpl4× at
132/(loxP)lacZ(loxP)-hGH and bi-transgenic mice
(n = 4/group). Crypts were scored by taking 35-mm slide
photographs of cecal wholemounts, obtained at × 37.5 magnification with a dissecting microscope, and projecting the slides
onto a 1-meter wide screen. In mice that only contained the Cre
reporter transgene, the percentage of X-Gal-positive crypts was
22.3 ± 2.9 (mean ± 1 S.D.; total number of crypts
surveyed = 66,686; n = 12,498-20,925
crypts/mouse). In bi-transgenic mice, only 2.2 ± 1.8% of crypts
were stained with X-Gal (total number of crypts surveyed = 84,009;
n = 16,073-23,772 crypts/mouse). The 90% reduction in
X-Gal-positive crypts noted between the two groups of mice is
statistically significant (p < 0.05 using Student's
t test).
The efficiency of recombination within each crypt was evaluated using
wholemount preparations and serial sections of cecum. As noted in the
Introduction, a hexagonal-shaped surface epithelial cuff surrounds the
orifice of each cecal and colonic crypt. Studies of chimeric mice have
shown that each epithelial cuff is monoclonal and represents the
cellular output of a single crypt (19, 27). We reasoned that if
recombination occurred in some, but not all, crypt epithelial cells
that support Fabpl4× at
132/(loxP)lacZ(loxP)-hGH expression, we should find
crypts with surface epithelial cuffs containing both lacZ-negative
(recombined) and lacZ-positive (unrecombined) cells. On the
other hand, if recombination were complete, then each cuff should be
monophenotypic. Monophenotypic crypts were the rule rather than the
exception. Surveys of wholemount preparations indicated that the
percentage of surface epithelial cuffs with mixed populations of
lacZ-negative and -positive cells was <0.1% in the cecums of
bi-transgenic animals (n = 4 animals; 2573-3504
lacZ-positive crypts scored/mouse). Surveys of X-Gal-stained serial
sections of cecum and colon also indicated that the loss of the
lacZ gene product from individual crypts was complete
(n = 6 mice).
The similar distribution of lacZ and hGH and the monophenotypic nature of the crypts suggest that recombination occurs over a period less than or equal to the time it takes cells to migrate from the base to the mid-portion of a crypt (~2-3 days) and/or that recombination is occurring in multipotent crypt stem cell(s), with the recombined reporter being distributed to all of its (their) progeny (see below).
Cre-mediated Recombination Occurs in All Cell Layers of the
Urothelium--
lacZ expression was completely eliminated from the
urothelium of bi-transgenic mice (Fig. 5, D and
F). Loss of lacZ was accompanied by the appearance of hGH
(Fig. 5H). Electron microscopic immunohistochemistry of
bladders harvested from bi-transgenic animals revealed hGH throughout
the urothelium, i.e. in the superficial facet (umbrella) cells (Fig. 6, A and
B), in the intermediate cell layers, as well as in the basal
layer (Fig. 6C). Immunoreactive hGH was not detected by EM
in the bladder epithelium of mice that contained only
Fabpl4× at 132/Cre or
Fabpl4× at
132/(loxP)lacZ(loxP)-hGH (e.g. Fig.
6D).
|
A System for Performing Inducible Gene Knockouts Confined to the Distal Small Intestinal, Cecal, and Colonic Epithelium
As noted above, mice expressing Cre recombinase under the control
of Fabpl4× at 132 initiate recombination of a
floxed locus in the intestine on or before E13.5. This early onset may
be a serious limitation to investigators who wish to examine the
function of a gene in the adult intestine, if loss of that gene's
product impairs or precludes normal gut development so that survival
beyond birth or weaning is not possible. It would be very useful to
have an inducible system that gave complete control over the timing of Cre-mediated gene inactivation, irrespective of the age of the mouse.
The inducible system we tested uses the "reverse"
tetracycline-controlled transactivator (rtTA). rtTA is a chimeric
protein consisting of a mutant E. coli Tn10 tetracycline
resistance operon repressor linked to the acidic activating domain of
herpes simplex virus VP16 (21). When the mutant rtTA binds tetracycline
analogs, such as doxycycline, it acquires the ability to bind to
tet operator sequences (tetO) and activate
transcription of ORFs placed under the control of tetO
linked to a minimal promoter (e.g. the human cytomegalovirus
immediate early gene 1 (IE1) promoter (PhCMV) (21)). Our plan was to create tri-transgenic mice that contained Fabpl4× at 132/rtTA, tetO
PhCMV-Cre, and a floxed recombination test locus, where
expression of Cre, and subsequent recombination of the target DNA,
would be entirely dependent upon administration of doxycyline (Fig.
7A). This system for inducing
Cre expression is different from a previously described system (29)
that used tetR/VP16 which is only active in the absence of tetracycline
(23, 30). In this latter system, Cre expression has to be suppressed by continuous administration of tetracycline until reaching the time during development or adulthood selected for induction of recombination (29).
|
The rtTA gene was placed upstream of nucleotide +3 of hGH in the same
Fabpl4× at 132/hGH vector used to generate
Fabpl4× at 132/Cre. As before, several
in-frame stop codons were present to block production of hGH from
rtTA-containing mRNA transcripts produced from this recombinant
DNA. Two pedigrees of FVB/N mice with this transgene were studied.
RNase protection studies of adult members of one pedigree established
that the distribution of rtTA mRNA was limited to their small
intestine, cecum, colon, and bladder. Unexpectedly, there was no
detectable bladder expression in adult members of another
Fabpl4× at 132/rtTA pedigree. This latter
pedigree was selected for further study because we wanted to create a
system where inducible Cre-mediated recombination would be restricted
to the intestine.
Fig. 7B is representative of results obtained from adult (6-40-week-old) transgenic mice from this line. The steady state level of rtTA mRNA increases progressively from the proximal to distal small intestine, is maximal in the cecum, and decreases in the colon. RNase protection assays of RNAs prepared from epithelial and mesenchymal fractions of their distal small intestine (see "Experimental Procedures") indicated that rtTA mRNA was confined to the epithelial compartment (Fig. 7, B-D). rtTA mRNA was not detected in any extra-intestinal tissues surveyed (Fig. 7B and data not shown).
Seven pedigrees of FVB/N tetO-PhCMV-Cre
transgenic mice were analyzed next. Members of each pedigree were
crossed to mice containing (loxP)hygroR(loxP).
Bi-transgenic mice generated using members of two of the tetO-PhCMV-Cre pedigrees showed no recombination
at (loxP)hygroR(loxP). Tri-transgenic mice were
then produced using members of one of these
tetO-PhCMV-Cre lines where basal Cre production
is undetectable, the Fabpl4× at 132/rtTA
pedigree that exhibited intestine-specific rtTA expression, and the
(loxP)hygroR(loxP) Cre reporter strain.
Four- to 10-week-old tri-transgenic mice were divided into two groups. An experimental group was allowed access to drinking water containing 2 mg/ml doxycycline and 5% sucrose for 4 days. (Oral administration of the inducer is noninvasive and allows rapid cessation of treatment.) A control group was given access to the sucrose solution minus doxycycline. Animals in both groups were sacrificed at the conclusion of this 4-day period.
PCR analysis of total cellular DNA prepared from 10 tissues revealed
that recombination in the experimental group was confined to the
intestine in the expected proximal-to-distal distribution (Fig.
8A). In contrast, the control
group had no detectable recombination at
(loxP)hygroR(loxP) in their gut or any other
tissues (Fig. 8A) (n = 2-3
mice/group/experiment; 2 independent experiments). Additional control
experiments, using bi-transgenic mice that only contained
Fabpl4× at 132/rtTA and
(loxP)hygroR(loxP) or
tetO-PhCMV-Cre and
(loxP)hygroR(loxP), showed no detectable
recombination after 4 days of doxycycline. Together, these controls
confirmed that recombination in tri-transgenic animals reflected
doxycycline-dependent rtTA transactivation of tetO-PhCMV-Cre.
|
PCR assays of colonic and cecal epithelial and mesenchymal DNAs prepared from doxycycline-treated tri-transgenic mice verified that recombination was absent from the mesenchyme (e.g. Fig. 8, B and C). This result is consistent with RNase protection studies that showed that rtTA mRNA transcripts were present in the epithelium and undetectable in the mesenchyme (Fig. 7B and data not shown).
Evidence That Recombination Occurs in Epithelial Cells with a Long Residence Time in the Crypt-- Although the induction of recombination was rapid enough to occur in a time frame equal to, or less than, one cycle of renewal for each of the intestine's epithelial cell types, we wanted to know how long the recombined allele would persist after removal of doxycycline. Therefore, groups of tri-transgenic mice were given access to water with doxycycline plus sucrose, or sucrose alone, for 4 days. Both groups were then switched to water without any supplements for an additional 10-60 days. Animals were sacrificed, and total cellular DNA was prepared from 8 tissues for PCR analysis of recombination. 0/6 tri-transgenic mice exhibited recombination when doxycyline was omitted from their drinking water. 5/6 mice manifested recombination in their intestine immediately after treatment, 4/6 after 10 days without drug, 4/4 after 20 days, and 3/5 after 30 days without doxycycline (2-3 mice/experiment; n = 2 independent experiments). The recombined allele can persist for 60 days (Fig. 8D). These results suggest that the induced Cre-mediated recombination occurs in an epithelial progenitor cell population with a long residence time in the crypt.
To examine the cellular patterns of recombination, tri-transgenic mice
containing Fabpl4× at
132/(loxP)lacZ(loxP)hGH as the Cre reporter, rather
than (loxP)hygroR(loxP), were treated with
doxycycline or vehicle alone, for 4 days. PCR analysis of total
cellular DNA prepared from ileum, cecum, and colon established that
recombination occurred only after doxycyline administration.
Immunohistochemical surveys of sectioned cecal crypts from both groups
of mice disclosed that recombination was associated with the appearance
of hGH-positive epithelial cells throughout the upper portions of
crypts and in their associated surface epithelial cuffs (Fig.
8E). These hGH-positive crypts were distributed throughout
the cecum as multi-crypt patches or as isolated crypts. Since the
Fabpl4× at 132/(loxP)lacZ(loxP)hGH
reporter is expressed in a similar patchy fashion, we were unable to
determine whether a hGH-negative cecal or colonic crypt in a
doxycyline-treated tri-transgenic mouse represented a failure to induce
Cre-mediated recombination in that crypt or the absence of
Fabpl4× at 132/(loxP)lacZ(loxP)hGH
expression. As with floxed hygroR, recombination
persisted after doxycyline treatment ended. For example, Fig.
8F shows hGH-positive crypts in tri-transgenic mice that had
been treated with doxycycline for 4 days followed by 10 days of water
without drug.
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DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Our studies indicate that transcriptional regulatory elements from
a fatty acid-binding protein gene (Fabpl4× at
132) can be used to engineer persistent Cre-mediated
recombination of a floxed target in the self-renewing adult mouse
intestinal epithelium. Two systems for performing Cre-mediated
recombination are described. In the first system, Cre expression is
placed directly under the control of Fabpl4× at
132. Recombination in the intestine is initiated as early as
E13.5, well before completion of its morphogenesis, and continues
throughout adulthood. This system also permits the effects of
Cre-directed recombination of a floxed allele to be evaluated in the
multilayer transitional epithelium of the renal calyces and pelvis,
ureters, and bladder as well as in the simple (one cell layer thick)
columnar epithelium of the intestine. The second system is more
elaborate and allows recombination to be induced at any time during
adulthood and only in the intestinal epithelium. The inducible system
uses Fabpl4× at 132-directed expression of a
reverse tetracycline-dependent transactivator (21) to
control Cre expression in a progenitor cell population having a long
residence time in intestinal crypts.
System 1, Fabpl4× at 132-directed Expression of Cre
Recombinase in the Intestinal Epithelium and Urothelium
Recombination in the Intestinal Epithelium--
This system allows
constitutive expression of Cre in the intestinal epithelium. We have
not been able to define the precise number of Cre-expressing crypts in
adult mice. This is because our Cre reporter strain of mice only
support expression of the unrecombined reporter transgene in 25% of
their cecal and colonic crypts. Nonetheless, our results suggest that
more crypts support expression of Fabpl4× at
132/Cre than expression of the reporter. If the fraction of
Cre-expressing crypts were equivalent to the fraction that
expressed the Fabpl4× at
132/(loxP)lacZ(loxP)-hGH reporter, then
the chance that an individual crypt would express both transgenes in
bi-transgenic animals would be (0.25)(0.25) = 0.0625, or 1 in 16, assuming that expression of each transgene is independent of the other.
We found that bi-transgenic mice containing Fabpl4× at
132/Cre and Fabpl4× at
132/(loxP)lacZ(loxP)-hGH had a 90% reduction in
the number of lacZ-positive crypts compared with age-matched
Fabpl4× at
132/(loxP)lacZ(loxP)-hGH controls. This value is
considerably greater than the 6.25% reduction we would predict by
simply assuming that an equivalent fraction of crypts independently
support expression of each transgene. Our analysis also indicated that,
in the vast majority of cases, when lacZ is lost from a crypt due to
recombination of the reporter transgene, it is lost from all of the
epithelial cells of the crypt. If Cre were not directed to all active
stem cells in a crypt, it would seem likely that the crypt would be populated, within its lifespan, by a mixture of cells, some with and
some without the recombined DNA.
Mice containing both the Fabpl4× at
132/Cre and Fabpl4× at
132/(loxP)lacZ(loxP)-hGH transgenes did not lose
all of their lacZ-positive cecal and colonic crypts. This indicates
that a subset of their crypts does not express Cre at levels sufficient
to support recombination. The size of this subset will have to be
defined once a Cre reporter strain is identified that supports
constitutive expression of a readily scored recombination marker
throughout the adult intestinal epithelium. However, the presence of a
cohort of Cre-negative crypts may provide a valuable internal reference
control for defining the effects of Cre-mediated gene inactivation. The
intestinal ecosystem exhibits complex regional differences in the
differentiation programs of its epithelial lineages, in the composition
of its microflora, and in its mucosal immune system (reviewed in Ref. 31). An in vivo system for defining the effects of loss of
the function a gene should, ideally, allow comparison of two juxtaposed populations of crypts, one populated by epithelial cells homozygous for
the wild type allele and another populated by cells homozygous for a
null allele. These juxtaposed crypts would experience a similar
microenvironment in a single animal and, thus, would provide a more
accurate way of defining the effects of loss-of-function than
comparisons made between wild type and knockout animals. Since
pathology in the human colon typically appears first in focal areas,
such a system could simulate the interactions that occur at the
interface between normal and abnormal cellular populations during
pathogenesis. When engineering gene knockouts in the distal intestine
using Fabpl4× at 132/Cre, the challenge will
be to devise a system to identify this valuable subset of reference
control crypts that do not support recombination.
Recombination in the Urothelium--
Prior to this study, we had
not appreciated that Fabpl4× at 132 was
active in the urothelium. Our initial characterization of the
properties of Fabpl4× at 132 in FVB/N
transgenic mice had used hGH as the reporter (11). We had noted that
total cellular RNA prepared from the kidneys of adult mice contained
low levels of hGH mRNA (11). Since immunoreactive hGH was
detectable in the proximal tubular epithelium of nephrons, we assumed
that these cells were the site of hGH mRNA production (11). Based
on the results obtained in the present study, this appears to have been
an erroneous conclusion. The presence of hGH in proximal tubular
epithelium was likely due to reabsorption of the protein from the
glomerular filtrate rather than synthesis of the protein.
Fabpl4× at 132/Cre allows the effects of
targeted gene inactivation to be compared in two distinct epithelia. In
contrast to the gut epithelium, turnover of the urothelium is very
slow: estimates in the normal adult mouse bladder range from months to
over a year (32, 33). The adhesive challenges faced by the multilayer urothelium differ from the challenges faced by the intestinal epithelial monolayer. The intestinal epithelium must maintain tight
cell-cell contacts to function as an effective biological barrier, yet
it must also support rapid cell migration over a substratum. The
urothelium must be able to stretch its surface area to accommodate
marked changes in urinary volume. It must also maintain one of the most
impenetrable barriers in the body. The urothelium's superficial facet
(umbrella) cells are able to establish and maintain this barrier, in
part because they possess high resistance junctional complexes that
block paracellular ion fluxes (34-36).
Fabpl4× at 132/Cre expression in the
urothelium of our pedigree of transgenic mice occurs as early as E16.5.
This feature will allow examination of the effects of targeted gene
inactivation during critical phases of urothelial morphogenesis (37,
38). In addition, our EM immunohistochemical studies demonstrate that
Fabpl4× at 132 can be used to direct Cre and
reporter gene expression to all layers of the urothelium. Several
studies have used nucleotides 3600 to +42 of the mouse uroplakin II
gene to direct expression of foreign gene products to the urothelium of
transgenic animals (39-41). Expression of these transgenes appears to
be restricted to suprabasal cell layers.
System 2, Inducible Expression of Cre Recombinase in the Intestinal Epithelium
As noted above, Fabpl4× at 132/Cre
expression is initiated as early as E13.5, before crypt morphogenesis
begins. Therefore, Fabpl4× at
132/Cre-mediated inactivation of genes essential for
intestinal development may not allow animals to survive until crypt
formation is completed during the third postnatal week (42). In
contrast, doxycycline-inducible, rtTA-regulated Cre expression allows a
floxed target gene to be disrupted in the intestinal epithelium at any
point during adulthood.
The two pedigrees of mice selected to assemble this inducible system
are distinctive for the following reasons. First, adult members of the
Fabpl4× at 132/rtTA pedigree only express the
reverse tetracycline-controlled transactivator in their small
intestinal, cecal, and colonic epithelium. Urothelial expression is
undetectable in this pedigree, unlike another Fabpl4×
at 132/rtTA pedigree we examined. Second, the
tetO-PhCMV-Cre pedigree had its transgene placed
at a genomic site where it is silent in the absence of rtTA and/or
doxycycline and is actively transcribed in the presence of rtTA plus
doxycycline. Bi-transgenic mice generated from crosses of the two
pedigrees have no discernible background of Cre expression in their gut
epithelium in the absence of doxycycline and functionally significant
expression when the doxycycline inducer is
administered.3
Doxycycline-induced recombination of a readily scored target locus can be used to mark crypt epithelial progenitors and their progeny. The residence time of progenitors in crypts can then be inferred by noting how long the marker (recombined locus) is represented in the amplified progeny of the progenitor after doxycycline is withdrawn. By using this approach, our results indicate that recombination occurs in progenitors that are subsequently maintained in distal small intestinal, cecal, and colonic crypts for at least 60 days. This 60-day period represents over half of the estimated 110-day lifespan of a crypt (see Introduction). This finding is consistent with the results of a recent clonal analysis of intestinal epithelial progenitors that used chemical mutagenesis to mark random cells by somatic mutation of the Dlb-1 gene. The clonal analysis indicated that pluripotent crypt progenitors are retained for several months (18).
Two points need to be clarified with this inducible system. First, as
with the constitutive Fabpl4× at 132/Cre
expression system, we must await identification of a Cre reporter
strain with robust and generalized expression in the adult gut
epithelium to determine what fraction of small intestinal, cecal, and
colonic crypts support recombination during and after administration of
doxycyline. Second, our PCR studies indicate that the distribution of
recombination in the small intestine varies somewhat between animals,
with the proximal boundary ranging from the proximal third (duodenum)
to the distal third (ileum). These variations cannot be correlated with
the length of time that transpires between withdrawal of doxycyline and
sacrifice of the mouse. We know that rtTA expression is lower in the
proximal compared with the distal half of the small intestine. The
variation in efficiency of recombination within the proximal small
intestine could be due to animal-to-animal differences in rtTA
expression, the dose of doxycycline received, or to other unknown
factors. At a minimum, once a better Cre reporter line is available,
additional studies will have to be conducted to test the effects of
different routes of doxycycline administration and different dosing schedules.
The price that must be paid for using a system where induction of
recombination is achieved by ligand-regulated rtTA transactivation of
Cre is more complex animal husbandry. Both Fabpl4× at
132/rtTA and the tetO-PhCMV-Cre will
have to be introduced into mice that are homozygous for the floxed
allele (or who have a floxed allele and a null allele of the gene of
interest). New Cre recombinases are being described whose enzymatic
activity depends upon an administered ligand (e.g. Refs.
43-46). Fabpl4× at 132-directed expression
of these types of Cre would simplify the task of generating mice where
gene knockouts could be induced in the gut epithelium. However, it
remains to be seen whether a system based on these Cre derivatives will
allow a "zero" background of recombination in the absence of
inducer or what the efficiency of induction will be.
Despite the complexity of the inducible system described in this
report, it should facilitate definition of gene function in the
intestine at selected times in pre- or postnatal life and under
selected physiologic or pathophysiologic conditions.
| |
ACKNOWLEDGEMENTS |
|---|
We thank David O'Donnell, Maria Karlsson, and Jason Himrod for invaluable assistance in generating and maintaining transgenic mice; Lisa Roberts for EM immunohistochemistry; and Melissa Wong, Lora Hooper, and Thad Stappenbeck for their support and suggestions throughout the course of this work.
| |
FOOTNOTES |
|---|
* This work was supported in part by Grants DK39760 and DK30292 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a National Research Service Award Post-doctoral
Fellowship DK09724 from the National Institutes of Health.
§ To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7047; E-mail: jgordon@molecool.wustl.edu.
2
The absence of expression in the small intestine
contrasts with other Fabpl4× at 132/reporter
transgenes, including Fabpl4× at 132/Cre, and
may be due to an insertion site effect, and/or to undefined cis-acting
elements present in the lacZ DNA.
3
The Cre ORF in the
tetO-PhCMV-Cre transgene is flanked by FRT
sequences recognized by Flp recombinase and is positioned immediately upstream of an intron and polyadenylation signal from the human -actin gene. Thus, the genomic locus where
tetO-PhCMV-Cre has inserted may be useful for
rtTA-induced expression of other gene products, i.e. the Cre
ORF could be excised by FLP recombinase and replaced with other open
reading frames.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
bp, base pair;
kb, kilobase pair;
PCR, polymerase chain reaction;
E, embryonic day;
P, postnatal day;
Fabpl4× at 132, nucleotides 596 to +21 of the rat Fabpl gene with four
additional copies of its nucleotides 177 to 133 inserted at
nucleotide 132;
hGH, human growth hormone;
ORF, open reading frame;
X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside;
PBS, phosphate-buffered saline;
PLP, periodate-lysine-paraformaldehyde;
Cy3, indocarbocyanine;
rtTA, reverse tetracycline-controlled
transactivator.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Cheng, H. (1974) Am. J. Anat. 141, 481-501[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Cheng, H. (1974) Am. J. Anat. 141, 521-535[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 461-479[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 503-519[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 537-561[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Wright, N. A., and Irwin, M. (1982) < |
