A reserve stem cell population in small intestine
renders Lgr5-positive cells dispensable
Hua Tian1, Brian Biehs2, Søren Warming1, Kevin G. Leong3, Linda Rangell4, Ophir D. Klein2& Frederic J. de Sauvage1
The small intestine epithelium renews every 2 to 5 days, making it
one of the most regenerative mammalian tissues. Genetic inducible
pools in this tissue. One pool consists of columnar Lgr5-expressing
cells that cycle rapidly and are present predominantly at the crypt
base1. The other pool consists of Bmi1-expressing cells that largely
reside above thecrypt base2. However,the relativefunctionsof these
two pools and their interrelationship are not understood. Here we
specifically ablated Lgr5-expressing cells in mice using a human
diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus.
We found that complete loss of the Lgr5-expressing cells did not
perturb homeostasis of the epithelium, indicating that other cell
types can compensate for the elimination of this population. After
ablation of Lgr5-expressing cells, progeny production by Bmi1-
expressing cells increased, indicating that Bmi1-expressing stem
cells compensate for the loss of Lgr5-expressing cells. Indeed,
lineage tracing showed that Bmi1-expressing cells gave rise to
Lgr5-expressing cells, pointing to a hierarchy of stem cells in the
cells are dispensable for normal intestinal homeostasis, and that
in the absence of these cells, Bmi1-expressing cells can serve as an
evidence for the interrelationship between these populations. The
Bmi1-expressing stem cells may represent both a reserve stem
cell pool in case of injury to the small intestine epithelium and a
source for replenishment of the Lgr5-expressing cells under non-
Two types of stem cells have been described in the small intestine
based on location and cycling dynamics1–4. Fast-cycling stem cells
express markers including Lgr5, Cd133 (also known as Prom1) and
as crypt base columnar cells (CBCs), these slender cells populate the
crypt and villi within 3 days, and are interspersed among the Paneth
cells that support them7,8. Slower-cycling stem cells, marked by
enriched expression of Bmi1 or mouse Tert (mTert), represent a rarer
cells are crucial for crypt maintenance2.
To study the function of Lgr5-expressing cells, we replaced the first
codingexonofLgr5withtwodistinctcassettes.The firstconsisted ofa
dsRED-IRES-CreERT2 sequence to enable genetic lineage tracing
studies by tamoxifen (TAM)-inducible expression of Cre in Lgr5-
expressing cells (Supplementary Fig. 1a, Lgr5CreERallele). The second
producing a fusion protein. Consistent with previous reports1, one
cells in a mosaic fashion and led to generation of labelled progeny for
in Lgr5DTRmice functioned as a reporter for Lgr5 expression (Fig. 1a)
and also conferred diphtheria toxin (DT) sensitivity on CBCs.
Expression of EGFP in mice carrying the Lgr5DTRallele was detected
at the membrane of cycling CBCs in every crypt (Supplementary Fig.
1c–e, CBCs are marked by asterisks).
We next set out to test the effects of eliminating Lgr5-expressing
cells by administering DT to Lgr5DTRmice. Twenty-four hours after
DT administration, all EGFP-positive cells were depleted, including
CBCs (Fig. 1a, b, j, k, p, q). Loss of Lgr5-expressing cells was further
confirmedby theabsence of Lgr5 messenger RNA(Fig. 1d,e) and was
accompanied by extensive apoptosis at the base of the crypts, with
shedding of dead cells into the lumen (Fig. 1m, n).
After sustained DT exposure for 10 days, both the EGFP reporter
and Lgr5 mRNA were completely absent from the base of the crypts
(Fig. 1c, f and Supplementary Fig. 2) but, notably, crypt architecture
was comparable to controls (Fig. 1g, i, j, l). Proliferating CBCs were
mostly or entirely by Paneth cells (Supplementary Fig. 3a, b). The
extensive apoptosis detected 24h after DT treatment had significantly
decreased by day 10 (compare Fig. 1n with o) but was still detectable.
No increase in crypt fission after DT treatment was observed by hae-
matoxylin and eosin staining at any time point (Fig. 1g–i).
Because Lgr5-expressing cells have been proposed to have a critical
roleinrenewaloftheintestine,it wassurprisingthatthe architectureof
the intestinal epithelium was essentially intact after ablation of Lgr5-
expressing CBCs (Fig. 1g–i). Within the villi, very little change in the
and goblet cells were abundant in the crypts and villi (Supplemen-
tary Fig. 3g, h, j). Upon CBC ablation, Paneth cells were found at the
bottom of the crypts and in some cases were mislocalized to the villi
of cells as assessed by BrdU pulse-chase labelling was normal (Sup-
plementary Fig. 4). The only major difference from controls that we
istration for 10days (Supplementary Fig. 3e, f, i).
Wedidnotdetectany Lgr5-expressingCBCsusingeitherthe EGFP
reporter or in situ hybridization after 10 days of DT (Fig. 1c, f and
Supplementary Fig. 2), but it was still possible that a few CBCs could
have escaped ablation and repopulated the epithelium, as a similar
scenario was reported in c-Myc and Ascl2 conditional null mice10,11.
To address this possibility directly, we visualized Lgr5-expressing cell
activity during DT selection by producing Lgr5DTR/CreER;R26R mice.
These mutant mice carried two null alleles at the Lgr5 locus, of which
one enabled ablation of Lgr5-expressing cells and the other enabled
lineage tracing of any possibly remaining Lgr5-expressing cells. These
mice died at postnatal day (P)1, consistent with previous reports that
Lgr5 null mice are not viable12. To analyse the postnatal gut, we grew
embryos under the kidney capsule of immunocompromised mice for
California 94080, USA.4Department of Pathology, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA.
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three weeks, at which point they formed crypts comparable to P17
intestine (Fig. 2a–e)13. After 10 days of TAM treatment, columns of
cells differentiated into all four major cell types of the intestinal
epithelium (Fig. 2a–e). Concomitant administration of DT and
TAM for 10 days eradicated all EGFP-positive CBCs (Fig. 2g), and
no cells descended from Lgr5-expressing cells were observed
(Fig. 2f), confirming that the Lgr5DTRalleleleadsto complete elimina-
tion of thesecells.Importantly,no abnormalitiesin graft morphology,
differentiation or proliferation were observed in these mice compared
to controls (Fig. 2a–j).
Although Lgr5-expressing cells were completely depleted within
24h of DT treatment,persistence of apoptoticbodies at the crypt base
throughout the 10-day DT treatment suggested that Lgr5-expressing
CBCs were continuously generated and eliminated during the treat-
ment (Fig. 1n, o). This notion was supported by the quick recovery of
Lgr5-expressing cells between 48 to 96h after the final dose of DT
(Fig. 1s–v). To follow the fate of the newly generated Lgr5-expressing
cells, mice implanted with Lgr5DTR/CreER;R26R embryonic intestine
fragments in the kidney capsule were allowed to recover for 6 days
in the presence of TAM following 6 days of DT treatment. A row of
cating that the newly formed Lgr5-expressing stem cells (Supplemen-
tary Fig. 5b, GFP-positive cells) gave rise to progeny that migrated
out of the crypt. When the converse experiment was performed by
Control 24 h DT 10 d DT
Cl. caspase 3
D6 D7D8 D10
Harvest (24 h) (96 h)
Figure 1 | Characterization of DT-mediated CBC ablation. a, EGFP is
detected on the membrane of Ki671proliferating CBCs in saline-treated
Lgr5DTR/1mice. b, One dose of DT eliminates all DTR–EGFP-positive cells at
cells.d–f, Lgr5mRNAisnormallypresentatthebottom ofthecrypts(d) and is
is intact after ablation of Lgr5-expressing CBCs. H&E, haematoxylin and eosin.
CBCs. m–o, Extensive apoptosisisobservedat thecrypt base 24h after DT and
tapers off by 10 days, but is still higher than controls. ‘Cl. caspase 3’ is cleaved
caspase 3. p–r, Electron microscopy shows that CBCs in controls are
characterized by slender nuclei and scant cytoplasm. No CBCs remain at the
granule-rich Paneth cells. TEM, transmission electron microscopy. s, Dosing
regimen for study of the recovery of Lgr5-expressing CBCs. t, No CBCs are
detected 24h after DT administration. D, day. u, A few Lgr51/Ki671CBCs
(arrows) recovered after 96h. Original magnification for panels: a–f, m–o and
t–v at 403; g–i at 203; j–l at 633; and p–r at 2,6503.
Lgr5DTR/CreER;R26R E15 kidney capsule
Paneth cellEndocrine cell
Figure 2 | Maintenance of normal crypt architecture is not mediated by
Lgr5-positive cells that have escaped ablation. a, Ten-day lineage tracing of
the base of the crypt in a grafted intestine piece from E15 Lgr5DTR/CreER
after loss of Lgr5 gene function. Lgr5-expressing stem cells can give rise to all
four major differentiated cell types (arrows). X-GAL-positive cells mark Lgr5-
positive stem cell progeny, which overlap with differentiated cell markers for
goblet(c), Paneth(d) andendocrinecell (e) lineages.PAS, periodic acid Schiff;
ChrA, Chromogranin A. f, Concurrent TAM and DT treatment kills all Lgr5-
expressing cells. No progeny of Lgr5-expressing cells (blue) are detected in the
grafted intestine. g–j, No GFP-positive cells are detected but proliferation and
differentiation are normal after DT-mediated ablation of Lgr5-expressing
CBCs. Original magnification for panels: a, b, f, g at 403; c–e, h–j at 633.
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injecting TAM for 6 days and then dosing with DT from days 6 to 12,
blue cells were only present in the upper region of the villi (Sup-
plementary Fig. 5c), indicating that progeny of Lgr5-expressing cells
cells were no longer available (Supplementary Fig. 5d, absence of GFP
signal) to supply labelled (blue) progeny to replenish the epithelium.
To study the long-term effects of CBC ablation, we isolated crypts
from Lgr5DTR/1mice to perform in vitro crypt organoid cultures14.
DT, as indicated by absence of GFP expression, gave rise to organoids
with similar efficiency as wild-type controls (Supplementary Fig. 6a,
b). These could be passaged in vitro in DT for up to 2 months without
losing their ability to expand and proliferate. No Lgr5-expressing
(GFP-positive) cells were detected in organoid epithelium as long as
the organoids were maintained in medium containing DT (Sup-
medium, Lgr5-expressing cells reappeared at the bottom of crypt-like
structures within 3 days (Supplementary Fig. 6c, GFP-positive cells).
Because we found that Lgr5-expressing CBCs were dispensable for
crypt maintenance, we next asked whether Bmi1-expressing stem cells
were mobilized to compensate for the loss of the Lgr5-expressing stem
gene expression16. Bmi1-expressing GFP-positive cells were most com-
monly observed at positions 3 to 6 above the crypt base (Fig. 3a), con-
sistent with the Bmi1 mRNA expression pattern in the small intestine2.
Upon depletion of Lgr5-expressing CBCs in Lgr5DTR/1;Bmi1GFP/1mice
increased three fold (Fig. 3a–d and Supplementary Fig. 7a), and the
cells increased by 40% compared to control animals (Supplementary
Fig. 7b). Of note, 55% of the total number of GFP-positive crypts in
(Fig. 3d and Supplementary Fig. 7b), compared with only 22% in
To trace the fate of cells descended from Bmi1-expressing cells
after elimination of Lgr5-expressing CBCs, we generated a Bmi1CreER
bacterial artificial chromosome (BAC) transgenic allele (Supplemen-
tary Fig. 8). Labelling kinetics using the Bmi1-CreER transgenic line
crossed with the R26R reporter were identical to previously reported
results using the Bmi1CreERknock-in allele2(Fig. 3f). Bmi1-
CreER;R26R;Lgr5DTR/1animals were treated with alternating doses of
are most abundant in the first 5cm of the duodenum, we focused our
expressing cells (Supplementary Fig. 7a), the proportion of LacZ-
positive crypts (either partially or fully labelled) also increased 34%
upon loss of Lgr5-expressing CBCs (Supplementary Fig. 7c). The most
of crypts were fully labelled in Bmi1CreER;R26R control mice during a
6-day lineage tracing period, which was comparable with previous
CBCs, the number of fully labelled crypts increased approximately 15-
the absence of Lgr5-expressing cells, Bmi1-expressing cells are capable
of directly giving rise to all intestinal cell types without going through
Lgr5-positive intermediate cells. However, Bmi1-expressing stem cells
(Fig. 3f, g), indicating thatalternativestemcellpools must compensate
for the loss of Lgr5-expressing stem cells in distal regions of the gut.
Lastly, we tested whether Bmi1-expressing cells give rise to Lgr5-
expressing cells under normal conditions. Because Bmi1- and Lgr5-
expressing cells represent distinct although overlapping cell popula-
tions, we carried out a series of short-term pulse-chase experiments
using Bmi1-CreER;R26R;Lgr5DTR/1mice. Twenty-four hours after
appeared as individuals, reflecting the normalpattern of Bmi1 expres-
sion (Fig.4a)intheinitially labelledcells.Bmi1-expressing cells(b-gal
positive) overlapped with Lgr5-expressing cells (GFP positive)
between positions 1 to 6 at the crypt base; the double-positive cells
peaked at positions 3 and 4 (Fig. 4a–c). This observation is consistent
tative polymerase chain reaction (qPCR) analysis) was readily detect-
able in Lgr5-positive cells11. Later, between 48–72h, clonal expansion
DuodenumJejunum IleumColonDuodenum Jejunum IleumColon
D0D2 D4 D6
Figure 3 | Bmi1-expressing stem cells are mobilized to compensate for the
loss of Lgr5-expressing CBCs. a, Rare Bmi1-expressing cells (arrows) are
detected at positions 3 to 6 of the crypt base in the duodenum of Bmi1GFP/1
ablation of Lgr5-expressing CBCs. c, Higher magnification showing a Bmi1-
up view of a crypt with multiple Bmi1-expressing cells after ablation of Lgr5-
expressing cells. Arrows in a–d indicate (Bmi1)-expressing cells. e, Dosing
regimen for lineage tracing of Bmi1-expressing cell progeny after ablation of
Lgr5-expressing CBCs. H, harvest. f, g, Whole-mount X-GAL staining of the
distal intestine. h, i, Close-up view of X-GAL-positive crypts in duodenum.
Most of the labelled crypts have less than five X-GAL-positive cells in Bmi1-
CreER;R26R control animals. Ablation of Lgr5-expressing CBCs stimulates
production of progeny by Bmi1-expressing cells. 36% of the crypts in the first
5cm of duodenum now become fully labelled (marked by arrows). Original
magnification for panels: a–d at 403; f, g at 1.23; h, i at 203.
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fromBmi1-expressing cells was evident, as b-gal/GFP double-positive
of 500 crypts at each time point and found that although a few cells
were b-gal/GFP double positive (that is, expressing both Bmi1 and
Lgr5) at 24h after TAM induction, this number doubled at 48h
(Fig. 4j, k). Similarly, lineage tracing from Bmi1-expressing cells
carried out in mice treated for 6 days with DT and allowed to recover
Together, these data show that Bmi1-expressing cells can give rise to
Lgr5-expressing cells both under normal physiological conditionsand
after insults that deplete CBCs. Similar to our observation, mTert-
expressing stem cells could also give rise to Lgr5-positive cells over a
5-day lineage tracing period9.
24 h TAM
48 h TAM
72 h TAM
D0D2 D4D6 D7D8D9
TMDT TM DTDT TM
72 h recovery
24 h TAM β-gal+ cell crypt position
12345678 9 10 11 12 13 14 15
Number of β-gal+ cells
Number of β-gal+ cells
48 h TAM β-gal+ cell crypt position
345678 9 10 11 12 13 14 15
Figure 4 | Bmi1-expressing cells give rise to Lgr5-expressing CBCs under
normal and injury conditions. a–c, Bmi1-CreER;R26R;Lgr5DTR/1mice were
dosed with 5mg TAM and harvested 24h later. b-Gal-positive cells (red)
derived from Bmi1-expressing cells overlap with Lgr5-expressing CBCs (GFP-
(red) show overlapping expression (marked by arrow) with Lgr5-expressing
stem cells is now evident by a streak of b-gal-positive cells migrating upward
(red). b-Gal-positive clones at lower crypt positions overlap with Lgr5-
expressing CBCs (arrow). j, k, Distribution of the Bmi1-positive stem cell
progeny (b-gal1cells) within the crypt at 24 and 48h after TAM induction.
j, Bmi1-expressing cells appear as singles throughout the crypt base between
positions 1 to 15. k, More cells are derived from Bmi1-expressing stem cells at
48h. A significant portion of b-gal1cells also express Lgr5 (GFP1, green
4 or 5. l, Dosing regimen used to study the recovery of Lgr5-expressing CBCs
from Bmi1-positive cells. m–o, Bmi1-positive cells give rise to a fully labelled
crypt (red), including newly formed Lgr5-expressing CBCs (GFP1, arrows).
The original magnification for all panels is 403.
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of the small intestine: an actively proliferating stem cell compartment
responsible for the daily maintenance of the intestine epithelium that is
characterized by the expression of Lgr5, Ascl2 and Olfm4 (refs 1, 11, 17)
and a distinct pool of stem cells expressing Bmi1. Our results lend
approaches18, and provide experimental evidence for recent models
predicting that theintestinecouldfullyrecoveraftercomplete elimina-
tion of cellular subpopulations deemed to be functional stem cells19.
are exclusively a subset of Lgr5-expressing cells11; rather they indicate
that under normal circumstances, Bmi1-positive stem cells are
upstream of rapidly cycling, Lgr5-expressing stem cells and replenish
the pool of active stem cells, either to avoid exhaustion of actively
cycling stem cells or to prevent the accumulation of damaged cells
that may lead to the development of tumours. Importantly, we also
presumably as a compensatory mechanism. Under these conditions,
Bmi1-expressing cells contribute directly to the generation of all cell
types of the intestinal epithelium to produce a functional organ until
it has been proposed that Bmi1-expressing stem cells are quiescent2,
this remains to be conclusively demonstrated.
Distinct stem cell pools with differing cycling dynamics have previ-
ously been observed in the hair follicle and in blood, organs that, like
the intestine, undergo regular bouts of proliferation and regenera-
lations of stem cells, and the precise hierarchical relationships among
that loss of Lgr5-positive cells is sustainable under short-term condi-
tions in vivo, it remains to be determined whether such a scenario can
persist for longer periods of time. Interestingly, depletion of Paneth
can be tolerated by mice for over 6 months without significant struc-
can function normally in the absence of CBCs. It will be important to
determine how different stem cell populations sense the activity of
other populations, whether rapidly cycling cells can repopulate more
quiescent stem cell populations, and whether additional subpopula-
tions of stem cells exist.
in Methods. Bmi1GFP/1mice were provided by I. Weissman16. All studies and
procedures involving animal subjects were approved by the Institutional Animal
Care and Use Committees of Genentech and the University of California, San
Francisco, and were conducted strictly in accordance with the approved animal
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 19 April; accepted 1 August 2011.
Published online 18 September 2011.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We gratefully acknowledge efforts by all the members of the
Genentech mouse facility, in particular R. Ybarra and G. Morrow. We are grateful to
N.Strauli,D.-K.Tran and A. Rathnayake for assistance with mouse breeding. We thank
M. Roose-Girma, X. Rairdan and the members of the embryonic stem cell and
members of the F.J.d.S. laboratory for discussions and ideas. This work was funded in
part by the National Institutes of Health through the NIH Director’s New Innovator
Award Program, 1-DP2-OD007191 and by R01-DE021420, both to O.D.K.
and collected data. H.T., B.B., O.D.K. and F.J.d.S. designed experiments, analysed the
data and wrote the manuscript. O.D.K. and F.J.d.S. are joint senior authors. All authors
discussed results and edited the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financial interests: details
accompany the full-text HTML version of the paper at www.nature.com/nature.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to F.J.d.S. (firstname.lastname@example.org) or O.D.K. (email@example.com).
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METHODS Download full-text
Lgr5 and Bmi1 vector construction. The constructs for targeting the C57BL/6
Lgr5 locus and the Bmi1 BAC transgene were made using a combination of
recombineering, DNA synthesis and standard molecular cloning techniques25,26.
For Lgr5, a 7,213bp fragment (assembly NCBI37/mm9, chr10:115,020,315-
115,027,527)from a C57BL/6BAC(RP23library) was first retrieved intoplasmid
pBlight-TK25. To generate the DTR–EGFP KI vector for Lgr5, a DTR–EGFP-pA-
loxP-Neo-loxP cassette was synthesized (Blue Heron/Origene, DTR–EGFP
sequence was based on that described previously27, and inserted right after the
and splice donor of intron 1 (a 212bp deletion). To generate the CreERT2 KI
vector, a dsRed2-IRES-CreERT2-pA-Frt-neo-Frt cassette was synthesized (Blue
final vectors were confirmed by DNA sequencing.
The Lgr5 KI vectors were linearized with NotI and C57BL/6 C2 embryonic
stem cells were targeted using standard methods (G418-positive and gancyclovir-
and confirmed by sequencing of the modified locus. Correctly targeted embryonic
stem cells were transfected with a Cre or Flpe plasmid, respectively, to remove the
using standard techniques, and germline transmission was obtained after crossing
the resulting chimaeras with C57BL/6N females.
For Bmi1, a 210kb C57BL/6 BAC (RP23-181D14, assembly NCBI37/mm9,
ing. The BAC contains the Bmi1 locus and considerable 59 and 39 flanking
sequence. An IRES-CreERT2-pA-frt-Neo-frt cassette was synthesized (Blue
Heron/Origene) and inserted, using recombineering, 85bp 39 of the Bmi1 stop
codon (after position chr2:18,606,193). Neo was then removed by transforming
the modified BAC into arabinose-induced, SW105 cells28,29expressing the yeast
protein Flp. C57BL/6 transgenic mice carrying the modified Bmi1 BAC were
obtained using standard pronuclear microinjection methods30and characterized.
We analysed the Lgr5DTR/1mice at 24h after DT administration (50mgk21,
intraperitoneal injection, n53), at 10 days of DT treatment (50mgkg21every
other day for 10 days, n55), 48h recovery (n53) and 96h recovery time points
days due to severe liver toxicity apparently mediated by a subset of Lgr5-DTR–
EGFP-expressing hepatocytes. We analysed Bmi1CreER;R26R;Lgr5DTR/1at 24h
b-gal-positive crypts were scored per mouse.
Renal capsule explants. 3–5-mm small intestine pieces from E15 Lgr5DTR/CreER
embryos (n53) were grafted under the renal capsule of 6–8-week-old athymic
nu/nu mice and allowed to develop for 3 weeks. We treated Lgr5DTR/CreER;R26R
renal grafts with 10 days TAM (n55), 10 days DT/TAM (n55), 6 days DT
followed by 6 days TAM (n55) and 6 days TAM followed by 6 days DT
(n55). Some GFP expression was seen outside of the CBC region due to perdur-
every 48h through intraperitoneal injections.
TAM in corn oil through intraperitoneal injection.
Transmission electron microscopy. The tissues were fixed in 1/2 Karnovsky’s
dylate buffer, pH 7.2), washed in the same buffer, and post-fixed in 1% osmium
by propylene oxide and embedded in Eponate 12 (Ted Pella). Thin sections were
JEOL JEM-1400 TEM.
Histology, immunohistochemistry and immunofluorescence. Animals were
perfused with 2% PFA. Small intestine and colon were flushed with 2% PFA
materials were paraffin embedded, sectioned at 3mm for histology and immuno-
GFP (Novus), chromogranin A (Neomarkers), b-gal (Cappel).
In situ hybridization and X-GAL staining. Full-length Lgr5 cDNA was cloned
into the pGEM vector to make anti-sense DIG-probe. Protocols for in vitro tran-
scription and in situ hybridization were as described previously31. Whole-mount
X-GAL staining was performed as described2.
Crypt organoid culture. Crypt isolation and culture were performed as
25. Warming, S., Rachel, R. A., Jenkins, N. A. & Copeland, N. G. Zfp423 is required for
normal cerebellar development. Mol. Cell. Biol. 26, 6913–6922 (2006).
26. Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based
method for generating conditional knockout mutations. Genome Res. 13,
27. Kissenpfennig, A. et al.Dynamics and function of Langerhans cells in vivo: dermal
dendritic cells colonize lymph node areas distinct from slower migrating
Langerhans cells. Immunity 22, 643–654 (2005).
highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33,
29. Lee, E. C. et al. A highly efficient Escherichia coli-based chromosome engineering
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