Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts.
ABSTRACT The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.
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ABSTRACT: Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16(-/-) ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16(-/-) ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.Nature Communications 05/2014; 5:3818. · 10.74 Impact Factor
Rapid tagging of endogenous mouse genes by
recombineering and ES cell complementation of
Dewang Zhou, Jin-Xiang Ren, Thomas M. Ryan, N. Patrick Higgins and Tim M. Townes*
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at
Birmingham, Birmingham, AL 35294, USA
Received July 16, 2004; Revised and Accepted August 20, 2004
ify endogenous genes in embryonic stem (ES) cells
time consuming. The timeline of an experiment from
the conception of an idea to the availability of mature
mice is at least 9 months. We describe a method in
which this timeline is typically reduced to 3 months.
Knockin vectors are rapidly constructed from bacter-
ial artificial chromosome clones by recombineering
followed by gap-repair (GR) rescue, and mice are
rapidly derived by injecting genetically modified ES
cells into tetraploid blastocysts. We also describe a
tandem affinity purification (TAP)/floxed marker gene
plasmid and a GR rescue plasmid that can be used to
TAP tag any murine gene. The combination of recom-
In recent years, protein–protein interactions in Saccharomyces
cerevisiae have been studied on a genome-wide level (1–4).
This large-scale approach is possible because endogenous,
protein-encoding genes in yeast are easily tagged with
sequences that facilitate protein complex purification. The
high efficiency of homologous recombination in this organism
permits modifications of endogenous genes with electro-
porated PCR products containing only 40 bp of homology.
The modified genes are expressed at normal levels; therefore,
physiologically relevant protein complexes are formed in vivo
and these complexes can be easily purified.
Similar studies have not been performed in mammalian
cells because the efficiency of homologous recombination is
several orders of magnitude lower. Two novel technologies
that have been developed in the past few years make
large-scale tagging of mammalian genes possible. The first
technology is recombineering. This method utilizes strains
of Escherichia coli that contain inducible recombination
(red) genes of bacteriophage l to carry out efficient,
homologous recombination between short, terminal homology
regions on a PCR product and sequences on a bacterial
artificial chromosome (BAC) (5,6). Several groups have
used this technology to construct standard, conditional and
knockin gene-targeting vectors for embryonic stem (ES)
cell modifications (7,8).
The second technology is ES cell complementation of tetra-
ploid embryos (9–11). This method allows mice to be cloned
directly from ES cells. F1 hybrid ES cells are injected into
tetraploid blastocysts and implanted into pseudopregnant
foster mothers. The animals that are born after 18 days are
derived totally from injected cells. If ES cells containing
tagged genes are injected, primary tissues could be used
directly for protein purification. In this paper, we combine
recombineering and ES cell complementation of tetraploid
blastocyts to produce knockin mice with a tagged allele in
3 months. The protocol is applicable to any mouse gene and
provides a method for large-scale tagging of mouse genes.
MATERIALS AND METHODS
The Erythroid Kruppel-like factor (EKLF) BAC DNA (75 kb)
was purified from the E.coli strain DH10B by the miniprep
method (12). Five hundred nanograms of this DNA was elec-
troporated into the E.coli recombineering strain DY380 and
chloramphenicol-resistant colonies were obtained. EKLF
BAC DNA prepared from DH10B and DY380 was digested
with EcoRI, KpnI and SalI and separated on agarose gels to
confirm that no DNA rearrangements occurred.
The TAPR plamid was constructed by assembling DNA frag-
ments containing tandem affinity purification (TAP) (1,13),
floxed PGK/Hyg (14) and KanR. The gap-repair (GR) plasmid
was constructed by inserting the MCI/TK gene (15) into
pBlueScript (Stratagene). The sequence of these two plasmids
and the plasmids themselves are available upon request.
Theprocedure forrecombineeringwasessentially asdescribed
previously (5). E.coli DY380 harboring the EKLF BAC was
grown at 30?C to an OD600 = 0.4–0.6 in Luria–Bertani
*To whom correspondence should be addressed. Tel: +1 205 934 5294; Fax: +1 205 934 2889; Email: firstname.lastname@example.org
Nucleic Acids Research, Vol. 32 No. 16 ª Oxford University Press 2004; all rights reserved
Nucleic Acids Research, 2004, Vol. 32, No. 16e128
Published online September 8, 2004
Medium (LB) in the presence of 20 mg/ml chloramphenicol.
The cells were then shifted to 42?C for 15 min to induce
expression of l recombination proteins Beta, Exo and Gam,
followed by chilling in ice water for 10 min. Electrocompetent
cells were prepared by washing the cells three times with 10%
glycerol. Five hundred nanograms of PCR products were
boiled for 1 min per 500 bp and then chilled in ice-cold
water. The denatured DNA fragments were electroporated
into 50 ml of ice-cold competent cells. After electroporation,
1 ml of LB medium was added to the cuvette, and the culture
was incubated at 30?C for 3 h with shaking. The cells were
then plated on selective media or pools of cells.
ES cell culture
V6.5 F1 hybrid ES cells (11) were maintained using standard
Tetraploid blastocyst complementation
HA–EKLF–TAP mice were produced by tetraploid embryo
complementation as described previously (10,11) with the
following modifications. Briefly, B6D2F1 female mice (The
Jackson Laboratory, Bar Harbor, ME) were hormonally ovul-
ated and bred to stud B6D2F1 males. During the following
morning, fertilized eggs were collected in M2 media (MR-
015-D, Specialty Media, Phillipsburg, NJ) and incubated over-
night at 37?C, 5% CO2in KSOM plus amino acids media
(MR-106-D, Specialty Media) to two-cell embryos. Two-
cell diploid embryos were fused individually to produce
one-cell tetraploid embryos using a 100 ms, 100 V pulse
from a CF-150/B electrofuser (Biological Laboratory Equip-
ments, Hungary) with hand-held, insulated electrodes in M2
media. Ninety-nine percent of the two-cell embryos were con-
verted to one-cell tetraploid embryos within 45 min by this
simple procedure.Tetraploid embryos were incubatedat 37?C,
5% CO2for 50 h to blastocysts in KSOM plus amino acids
media. Approximately 90% of tetraploid embryos produced
blastocysts with an excellent blastocoele cavity. The ES cells
were injected into tetraploid blastocysts with the aid of a Piezo
Primetech, Ibaraki, Japan). Injected tetraploid blastocysts
weretransferred tothe uterus ofpseudopregnant CD11female
recipients (Charles River Laboratories, Wilmington, MA).
Approximately 10% of tetraploid blastocysts that were
injected with genetically modified ES cells yielded live-
born pups. Forty percent of these pups survived to adulthood.
Approximately 25% of tetraploid balstocysts that were
injected with unmodified V6.5 ES cells yielded live-born
pups and 50% of these pups survived to adulthood.
The sequences of all the primers used in this paper are listed
AGCC-30; KLF+79, 50-AGTCCTCCTGGGTGTCCAGAA-30.
Primers for TAP tagging: KLF-L8-TAP-F2, 50-tcgtcccttctgct-
AGATG-3; EKLF–TAP-R5, 50-tataatggctcatcttttgggatacggtc-
CCTGCAGATAACTTC-30. Primers for GR: pBSTK-GAP-F,
ATGATTACGCCAAGC-30; pBSTK-GAP-R, 50-cacagcca-
ATTCCACACAAC-30. Primers for PCR screening: EKLF-F,
50-GAGAGAAGCCTTATGCCTGC-30; EKLF-R, 50-TCTG-
GTGGTCTATATTGCTG-30; KLF-188, 50-AGACGCACAC-
CACACACATA-30; HA-R, 50-AGTCGGGCACGTCGTAAG-30;
50-GCAGCAGCAGTAGTTTCATC-30; TAP-R2, 50-CGCC-
TGAGCATCATTTAG-30; ES-R1, 50-AGGGTCACCTAGT-
GCTTCCA-30; Kan-F2, 50-GCCTTCTTGACGAGTTCTT-30.
RESULTS AND DISCUSSION
The generation of knockin mice in 3 months is illustrated by
tagging the endogenous murine EKLF gene with HA and TAP
(1,13). EKLF is a C2H2zinc finger transcription factor that is
essential for the globin gene regulation (17–20). To study the
cellular localization of EKLF during development and to
define protein complexes containing EKLF in primary
mouse tissues, we inserted an HA tag at the 50end and a
TAP tag at the 30end of the endogenous mouse EKLF
gene. In this paper, we report the method utilized to produce
HA/EKLF/TAP knockin mice rapidly. The overall strategy is
illustrated in Figure 1.
An EKLF BAC clone in DH10B was obtained from the
BACPAC Resources Center at Children’s Hospital Oakland
Research Institute (CHORI). BAC DNA was purified and
electroporated into E.coli DY380, which harbors the l
phage red system (5,6). To insert a TAP tag (with an
8 amino acid linker) into the 30end of the EKLF gene, two
long oligonucleotides were used to amplify by PCR a
TAPR (TAP Recombineering) plasmid containing the follow-
ing cassette: linker/TAP/loxP-PGK/Hyg/KanR-loxP. The 50
79 or 77 bases of the two primers are homologous to sequences
either immediately upstream or immediately downstream of
the EKLF stop codon (TGA) (Figure 1) and the 3020 or
22 bases are homologous to sequences at the ends of the
TAPR cassette. The 3.9 kb PCR fragment was gel purified
and electroporated into DY380/EKLF BAC after the induction
of the l recombination system by a 15 min incubation at 42?C.
Thirty kanamycin-resistant colonies were obtained and PCR
with primers EKLF-F and EKLF-R (Figure 2A) was used to
identify homologous recombinants. As shown in Figure 2A,
the 4084 bp band that was predicted for homologous
recombinants was observed in four colonies. Twenty-one
colonies contained only a 372 bp band and, therefore, were
non-homologous recombinants. The remaining five clones
were mixtures of homologous and non-homologous recombi-
nants. Homologous recombinants were derived from these five
clones when the original EKLF BAC was lost after further
growth and isolation of single colonies. We then purified BAC
DNA from the nine homologous recombinants and sequenced
the TAP tag and the PGK/Hyg gene. Three of the nine BACs
contained the correct sequences.
An HA tag was inserted at the 50end of the EKLF gene in
one of these clones by recombineering. Two methods were
e128Nucleic Acids Research, 2004, Vol. 32, No. 16
PAGE 2 OF 7
used to prepare an HA DNA fragment for electroporation into
DY380 cells containing the TAP-tagged EKLF BAC clone.
First, a 202 bp fragment containing HA–(11 amino acid
linker)–EKLF sequences was derived by PCR amplification
of anHA–linker–EKLF plasmid (21). This fragmentcontained
an HA–linker sequence plus 60 bp of homology upstream and
79 bp of homology downstream of the EKLF ATG. Second, a
of homology upstream and downstream of the EKLF ATG
was derived by annealing two partially overlapping 98 base
oligonucleotides (48 base overlap) and by extending these
primers with T4 DNA polymerase. The fragments were
gel-purified and electroporated into DY380 cells containing
the TAP-tagged EKLF BAC clone. Correctly recombineered
BACs were identified by sib selection. Pools of 100 cells and
10 cells were prepared by serial dilution, expanded by
overnight growth and screened by PCR for homologous
recombinants using an EKLF upstream primer (KLF-188)
and a primer from within the HA tag (HA-R). Data for the
first method of HA tagging are shown in Figure 2A. All the
100-cell pools and 13 of the 32 10-cell pools were positive for
homologous recombinants. Four of the 10-cell pools were
streaked on plates and individual colonies were examined
by PCR. Four homologous recombinants were identified
and sequencing confirmed that three of these clones contained
the correct HA sequence. A similar efficiency of homologous
recombination was observed using the second HA tagging
method (data not shown).
The modified gene (HA–EKLF–TAP) with ?6.0 kb of
sequence upstream of HA and 1.0 kb of sequence downstream
TAPwasrescuedfromthe HA–EKLF–TAP BACclonebyGR
(Figure 1). The GR vector was a 5.0 kb DNA fragment gen-
erated by PCR amplification of a GR plasmid containing an
ampicillin-resistance gene, a ColE1 replicon and a pMCI/TK
gene (15). Two long primers were utilized for PCR. The 50
50 bases of the two primers were homologous to sequences
either 6.0 kb upstream of HA or 1.0 kb downstream of TAP
and the 3020 or 22 bases were homologous to sequences at the
ends of the linearized GR plasmid. The 5.0 kb PCR fragment
was gel-purified and electroporated into induced DY380
containing the HA–EKLF–TAP BAC. Nine ampicillin/
kanamycin-resistant colonies were picked and the DNA
prepared from these colonies was digested with NotI,
EcoRI, HindIII and XhoI. As shown in Figure 2B, the
restriction patterns of all nine clones indicated that the 18.3
kb plasmid contained correctly captured HA–EKLF–TAP
with the appropriate homology regions. Sequence analysis
of the junctions between HA–EKLF–TAP and the GR plasmid
vector in these clones confirmed that the predicted structure
was produced (data not shown).
To replace the wild-type EKLF gene with HA–EKLF–TAP
in ES cells, one of the capture plasmids was linearized by NotI
digestion and electroporated into hybrid B6/129 F1 ES cell
line V6.5 (11). After 8 days of selection in hygromycin and
gancylovir, eight ES cell colonies were obtained. Genomic
DNA from these colonies was analyzed by PCR with primers
Kan-F2 and ES-R1. As shown in Figure 3A (ES 30PCR),
colonies designated ES5 and ES7 (lanes 10 and 12) produced
a 1.6 kb band that is predicted for homologous recombination.
PCR amplification of HA–EKLF–TAP BAC DNA was used as
a positive control (lane 1), and PCR amplifications of linear-
ized capture plasmid (lane 2) and wild-type V6.5 ES cell DNA
(lane 3) served as negative controls.
Correct targeting at the 50end was assessed by PCR ampli-
fication of genomic ES cell DNA using a TAP primer (TAP-
R2) and a primer (KLF-9F2) derived from the sequence
upstream of the 6 kb homology (Figure 3A, ES 50PCR).
As expected, ES5 and ES7 (lanes 4 and 5) produced a
9.1 kb band which was identical to the one generated by
the HA–EKLF–TAP BAC DNA control (lane 1). A 9.1 kb
PCR fragment was not observed with wild-type V6.5 cell
DNA (lane 2). Southern blot analysis of ES5 and ES7 DNA
HA–EKLF–TAP in these cells (Figure 3B). Lanes 4 (ES5)
and 5 (ES7) of Figure 3B demonstrate that both ES cell clones
Linearization of Capture Plasmid
and Electroporation into V6.5 ES Cells
Figure 1. Overview of method. A BAC clone containing the EKLF gene was
transferred from the E.coli strain DH10B to the recombineering strain DY380.
Thesecells wereelectroporatedwithPCR products containinganHAtag anda
TAPR (Linker/TAP/loxP-PGK/Hyg/KanR-loxP) cassette flanked by 60–79 bp
EKLF homologies. Cells containing the correctly modified BAC were
electroporated with a PCR product containing AmpR, ColE1 and herpes
simplex virus (HSV) thymidine kinase sequences flanked by 50 bp
homologies. By GR, this fragment rescued a 13.3 kb fragment containing
the modified EKLF gene plus 6 kb of upstream sequence and 1 kb of
downstream sequence. The resulting 18.3 kb plasmid was linearized with
NotI and electroporated into the F1 hybrid (B6/129) ES cell line, V6.5.
homologous recombination by PCR and Southern blot hybridization.
Correctly targeted ES cells were subsequently injected into tetraploid
blastocysts to clone mice that are heterozygous for the tagged EKLF gene,
PAGE 3 OF 7
Nucleic Acids Research, 2004, Vol. 32, No. 16e128
contain the 4.1 kb wild-type allele and the 7.7 kb knockin
allele. Control lanes 1 and 2 are the wild-type and knockin
EKLF BAC DNA, respectively, and lane 3 is wild-type ES
Mice containing the HA–EKLF–TAP knockin allele were
rapidly produced by the method of tetraploid embryo comple-
mentation. The production of mice by this method precludes
the generation of chimeric mice that must be bred to demon-
strate germline transmission. ES5 and ES7 cells were injected
into tetraploid blastocysts and 121 of these blasts were trans-
ferred to the uteri of foster mothers. Twelve pups were born
alive andfivesurvived toadulthood.Allwereheterozygousfor
the HA–EKLF–TAP allele and no chimerism was observed.
Each animal was derived entirely from the modified ES cells.
All the steps described above were completed in 3 months.
To demonstrate that HA–EKLF–TAP is functional in vivo,
we derived mice that were homozygous for the knockin.
Heterozygous HA–EKLF–TAP males were bred to C57B6
females that express CRE recombinase from the cytomega-
lovirus promoter and the offspring were mated. Tail DNA
from one litter of six pups was analyzed by PCR with 50
primers KLF-188 and KLF+96, and the results are illustrated
Figure 2. HA- and TAP-tagging of the EKLF gene by homologous recombination in E. coli DY380. (A) Sib selection and kanamycin selection of HA- and TAP-
EKLF BAC clone. For the HA tag, pools of cells were prepared and screened by PCR with primers KLF-188 and HA-R. The predicted product for homologous
PCR with primers EKLF-F and EKLF-R. The predicted products for homologous recombinants is 4084 bp. A 372 bp product is predicted for the wild-type EKLF
BAC. (B) Mapping of GR plasmids. To confirm the capture of HA–EKLF–TAP with appropriate flanking sequence, GR plasmids were digested with NotI, EcoRI,
move as a single band; whereas the 6.1 and 5.7 kb bands in HindIII digestion are inseparable in this gel.
e128Nucleic Acids Research, 2004, Vol. 32, No. 16
PAGE 4 OF 7
in Figure 4A (lanes 4–9). One of six pups was homozygous for
HA–EKLF–TAP (lane 7). The six tail DNAs were also
analyzed by PCR with 30primers EKLF-F and EKLF-R
(lanes 6–11, Figure 4A). The segregation pattern was the
same as observed above, and the same pup was homozygous
for HA–EKLF–TAP (lane 9). Multiple litters were genotyped
and, as expected, one-fourth of the pups were homozygous for
HA–EKLF–TAP. Blood smears and hematological values
(data not shown) were normal in these animals; therefore,
HA–EKLF–TAP is functional in vivo.
Western Blot analysis of bone marrow cell extracts with an
anti-HA monoclonal antibody confirmed HA–EKLF–TAP
expression. Bone marrow cells from wild-type and HA–
EKLF–TAP homozygous mice were separated into Ter119+
and Ter119?fractions on a MACS column (Miltenyi Biotech)
using anti-Ter119 antibody linked to magnetic beads. Whole-
cell protein extracts from these cells were analyzed. As shown
in Figure 4B, two bands with molecular weights of ?65 and
70kDawere detectedinthe Ter119+bonemarrow cell fraction
from HA–EKLF–TAP mice (lane 5) but not in Ter119+cells
from wild-type mice (lane 3). Low levels of the HA–EKLF–
TAP doublet were also observed in the Ter119?fraction from
HA–EKLF–TAP mice (lane 4). Similar bands were detected in
a positive control sample derived from COS cells transfected
by PCR. For ES 50PCR, lane 1, modified EKLF BAC DNA; lane 2, wild-type V6.5 ES cells; lane 3, primer control; lanes 4 and 5, ES clones 5 and 7; lane M,
molecularweight marker.For ES 30PCR,lane 1, modifiedEKLFBACDNA; lane2, linearizedgap-repairedplasmidDNA;lane 3, wild-typeV6.5 ES cells; lane 4,
primer control; lane 5, 1.0 kb marker; lanes 6–13, ES cell clones 1–8. (B) Southern blot of targeted ES cell clones. Genomic DNA purified from ES cell clones was
digested with HindIII and analyzed by Southern blot hybridization. The probe was a 447 bp SnaBI fragment isolated from sequence downstream of the 1.0 kb
homology. Lane 1, wild-type EKLF BAC; lane 2, modified EKLF BAC; lane 3, wild-type V6.5 ES cells; lanes 4 and 5, ES5 and ES7.
PAGE 5 OF 7
Nucleic Acids Research, 2004, Vol. 32, No. 16e128
with a HA–EKLF–TAP cDNA clone, pDZ17 (lane 2), but no
protein wasdetectedinwhole-cell extracts preparedfromCOS
cells transfected with a control vector pSG5 (lane 1). These
results demonstrate that EKLF is expressed predominately in
Ter119+hematopoietic cells from the bone marrow.
The cellular localization of EKLF in bone marrow cells was
testedbyimmunofluorescence microscopy.Bonemarrow cells
were prepared from wild-type and HA–EKLF–TAP homozy-
gous mice and stained with PE-conjugated anti-Ter119 anti-
body. Ter119+cells were then purified by FACS sorting, and
stained with FITC-conjugated anti-HA monoclonal antibody.
Figure 4C illustrates fluorescent, digital sections of these cells.
The bottom left panel, which illustrates the merge of Hoechst,
anti-Ter119-PE and anti-HA-FITC stains, demonstrates that
HA–EKLF–TAP is localized in the nucleus in adult bone
marrow erythroid cells.
The combination of recombineering and tetraploid blastocyst
complementation (5,6,10,11) provides a method to produce
knockin mice in 3 months. The novel plasmids described in
this paper permit rapid TAP tagging of any gene in the murine
genome. We anticipate that the use of this strategy will enable
large-scale tagging of mouse genes and systematic identifica-
tion of protein complexes during development.
We thank Dr Don Court for the E.coli recombineering strain
DY380. We are grateful to Dr Rudolf Jaenisch for the V6.5 ES
cells and members of the Townes laboratory for helpful dis-
and T.M.T., and a NIGMS grant to N.P.H.
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