Mice Lacking Pituitary Tumor Transforming Gene Show Testicular and Splenic Hypoplasia, Thymic Hyperplasia, Thrombocytopenia, Aberrant Cell Cycle Progression, and Premature Centromere Division
Zhiyong Wang,
Run Yu and
Shlomo Melmed
Department of Medicine, Cedars-Sinai Research Institute, University
of California Los Angeles School of Medicine, Los Angeles, California
90048
Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Research Institute, Room 2015, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail:
melmed{at}csmc.edu
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ABSTRACT
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Tumorigenic pituitary tumor transforming gene (PTTG) is a
mammalian homolog of Xenopus securin that inhibits
chromatid separation, is overexpressed in many human tumor types, and
mediates transcriptional activation. Loss of yeast securin Pds1p or
Drosophila securin pimples is lethal. Here we
show that mice lacking PTTG (PTTG -/-) are, surprisingly, viable and
fertile; but they have testicular and splenic hypoplasia, thymic
hyperplasia, and thrombocytopenia. PTTG -/- mouse embryo
fibroblasts exhibited aberrant cell cycle progression with
prolonged G2-M phase and binucleated and multinucleated
nuclei with increased aneuploidy. PTTG -/- mouse embryo fibroblast
metaphases contained quadriradial, triradial, and chromosome breaks, as
well as premature centromere division. The results show that PTTG
functions to maintain chromosome stability, cell cycle progression, and
appropriate cell division. Moreover, mammalian sister chromatid
separation, an important transition in the cell cycle, is likely
regulated by mechanisms in addition to securin.
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INTRODUCTION
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CELL GROWTH RELIES on cell cycle
progression, requiring several regulators including p53, retinoblastoma
(Rb), cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors such
as p21 and p16 (1, 2). DNA synthesis, chromosome
segregation, spindle assembly, and cytokinesis all occur in ordered
sequence during the cell cycle. Loss or mutation of genes controlling
these processes leads to dysfunctions of cell cycle progression and
frequently to tumorigenesis and apoptosis. For example, mice lacking
p53 show unregulated G1 checkpoint control and a
high prevalence of spontaneous tumor development (3); mice
lacking Rb do not survive fetal development while Rb+/- mice developed
pituitary tumors at 8 months (4); mice lacking p21 undergo
normal development but show defective G1
checkpoint control (5). Recently, a family of proteins
including securins, separins, and cohesins were found to play important
roles in sister chromatid separation during M phase
(6) and exhibit characteristics of cell cycle
regulators. Securin proteins (Saccharomyces cerevisiae
Pds1p, Schizosaccharomyces prombe Cut2,
Drosophila pimples, Xenopus securin) reach
highest expression levels in M phase and share at least one destruction
box and a nine-amino acid consensus motif [RX(A or V or L) LGXXX N]
originally identified in B-type cyclins (7, 8, 9, 10). The
separins (Esp1p, Cut1, BimB) share a conserved carboxy-terminal
domain, which binds to securins (7, 8, 11). Securin
accumulation during interphase and their binding to separin prevents
premature separin activation. During a normal cell cycle, anaphase
promoting complex (APC) eventually degrades securin, thus activating
separin to facilitate chromosome segregation (6). In
this sense, securins function as inhibitors of chromatid separation
during anaphase. Thus far, mammalian securin or separin
characterization has been limited (9).
Pituitary tumor transforming gene (PTTG) has 44.6% amino acid
identity with Xenopus securin, and PTTG contains a
destruction box (RXLGXXXN) and cyclin B-like nine-amino acid consensus
motif (9). Originally isolated from pituitary tumor
GH-secreting cells by differential display, PTTG is considered a
protooncogene because PTTG overexpression in NIH3T3 cells induces cell
transformation and in vivo tumor formation
(12). Although abundant only in normal testis and thymus
(13), PTTG is highly expressed in various human tumors and
is responsive to E induction (14, 15). Moreover, PTTG
mediates promoter transcriptional activation (16) and
utilizes c-myc as its downstream gene target
(17). Indeed, PTTG preferentially localizes in the nucleus
(18), its expression levels change in a temporal pattern
during cell cycle progression peaking during M phase, and it is
phosphorylated by cdc2 and MAPK (19, 20).
Yeast securin Pds1p deletion mutants separate sister chromatids
inefficiently and are lethal at 37 C, but these mutants survive and
proliferate at 25 C with unaffected sister chromatid separation
(7). Drosophila securin pimples loss mutation
results in defective sister chromatid separation during mitosis,
defective cytokinesis, and recessive lethality (10). More
recently, inactivation of human securin (hsecurin) in a karyotypically
stable human colorectal cancer cell line resulted in a higher
chromosome loss rate in these cells while remaining viable
(21). To understand mammalian securin PTTG function, we
disrupted the murine PTTG gene by homologous recombination. We report
here that mice lacking PTTG are, in contrast to yeast or
Drosophila-lacking securin, viable; but they show testicular
and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant
cell cycle progression, chromosome instability, and premature
centromere division.
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RESULTS
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PTTG -/- Mice Are Viable and Fertile
In the targeting vector, part of exon I including the ATG start
codon, exon II, and exon III were replaced by a neomycin cassette (Fig. 1a
) resulting in a PTTG null
mutation, as determined in both Southern and Northern blot (Fig. 1
, b and c).

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Figure 1. Targeted Disruption of the PTTG Gene
a, Genomic structure of the murine PTTG gene and targeting construct.
Endogenous PTTG contains five exons, depicted as E1E5. A 4.2-kb
HindIIIEcoRI fragment of PTTG including
exons 2, 3, and part of exon 1 was replaced with a pGK-neo cassette.
The solid box designated "probe" represents the
region used for Southern blotting. b, Southern blot analysis of genomic
DNA derived from mouse tails with the indicated PTTG genotype. DNA was
digested with HindIII and probed with the labeled 350-bp
fragment shown in panel a. c, Northern blot analysis of total RNA
derived from mouse testis with the indicated PTTG genotype. Murine PTTG
exon 3 cDNA fragment was used as probe.
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Surprisingly, in contrast to the lethal phenotypes of yeast or
Drosophila lacking securin, PTTG knockout mice are viable
and fertile. Of the first 100 F2 progeny, 23 were
PTTG +/+, 51 were PTTG +/-, and 26 were PTTG -/-, suggesting that
PTTG deficiency did not result in significant intrauterine mortality.
Nonetheless, PTTG -/- mice demonstrate female subfertility,
e.g. in 15 pairs of breeding, the average litter size
derived from a PTTG -/- dam (35 pups per litter) was less than half
of the litter size from a PTTG +/- or +/+ dam (711 pups per litter)
(Table 1
).
PTTG -/- Mice Demonstrate Testicular and Splenic Hypoplasia,
Thymic Hyperplasia, and Thrombocytopenia
PTTG -/- mice had reduced testicle weight, reduced spleen
weight, and enlarged thymus (Table 2
,
Fig. 2a
), while ovarian weights did not
differ. Testicular hypoplasia was more severe in sexually mature
than in immature mice (Table 2
). The PTTG -/- adult testicle weight
is 4555% of PTTG +/+ animals. Splenic hypoplasia was apparent after
weaning and continued for up to 8 months observation time: PTTG -/-
spleen weight 5075% of PTTG +/+. Thymic hyperplasia was also
observed approximately 4 wk after birth and continued up to 8 months
observation, with PTTG -/- thymus weight 115135% of PTTG +/+.
Thymic hyperplasia was more pronounced at an early age (45 wk). PTTG
is abundantly expressed in normal testis and thymus, but not in spleen
or ovary (13). The weight changes observed in testis and
thymus thus suggest cell type differences in PTTG effects on cell
growth. In the presence of thymic overgrowth, and as the spleen is
composed mainly of T and B cells, reduced PTTG -/- spleen weight
suggests reduced B cell population and weakened antibody production.
This is supported by impaired IgM and IgG1a antibody production in PTTG
-/- mice when keyhole limpet hemocyanin is used as an
immunogen (unpublished data). In the thymus, thymic hyperplasia
is probably not due to reduced apoptosis, as PTTG -/- thymocytes
demonstrated similar in vitro responses to 20
nM dexamethasone or 3 grays (Gy) irradiation as
compared with wild-type (WT) thymocytes (data not shown), and similar
to GADD45a -/- mice with thymic hyperplasia despite functioning
thymocyte apoptosis mechanisms (22). Nevertheless, the
distribution of CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes differed
significantly after PTTG disruption (Fig. 2b
): CD4+CD8- thymocytes
represent approximately 13.5% of total PTTG -/- thymocytes
vs. about 6.5% in PTTG +/+ mice. Moreover, hematological
analysis showed that PTTG -/- mice are thrombocytopenic, despite
normal bone marrow megakaryocyte numbers. PTTG -/- platelet numbers
ranged from 4065% of PTTG +/+ mice, and PTTG -/- mice bleeding
time was prolonged (1619 min vs. 510 min in PTTG +/+
mice, P < 0.005) (Fig. 2c
).

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Figure 2. Phenotype Observations in PTTG +/+ and PTTG -/-
Mice
a, Photographs of testis, spleen, and thymus from PTTG +/+ and PTTG
-/- mice: testis samples from animals at 30 wk of age; spleen and
thymus samples from animals aged 45 wk. b, Relative distribution of
thymocyte subsets in the 5-wk thymus were determined by staining for
expression of indicated lineage-specific cell surface antigens and cell
sorting by flow cytometry. Relative percentages of cells exhibiting
each cell surface characteristic are indicated. c, Tail bleeding times
for PTTG +/+ and PTTG -/- mice at 8 wk. Each point represents one
individual mouse, and results were generated from two separate
experiments in 12 mice.
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PTTG -/- MEFs Exhibit Aberrant Cell Cycle Progression, Chromosome
Instability, and Premature Centromere Division
PTTG -/- and PTTG +/+ mouse embryonic fibroblasts (MEFs) both
derived at passage 3 demonstrated similar doubling times (
30 h) but
different cell cycle parameters (Fig. 3a
). The PTTG -/- MEF
G1 phase was shortened (10.1 vs.
18.2 h), with a prolonged G2-M phase (7.2
vs. 1.4 h), implying deficient
G0-G1 checkpoint control
and delayed progression of G2-M. PTTG -/- MEFs
showed a flow cytometric pattern similar to a pattern in DNA-damaged
cell population such as one that was observed in
-irradiated WT PTTG
+/+ MEFs (Fig. 3b
).

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Figure 3. Cell Cycle Analysis of PTTG +/+ and PTTG -/- MEFs
a, PTTG +/+ and PTTG -/- MEFs were plated at low (4 x
103/cm2), medium (8 x
103/cm2), and high (1.6 x
104/cm2) concentrations, respectively, and cell
doubling times and cell cycle parameters were assessed as previously
described (43 ). Doubling time determined in this
experiment was 30.6 h for WT and 29.8 h for PTTG -/- cells.
The length of time (hours) an average cell spends in the cell cycle
phases is indicated. b, Flow cytometry analysis of PTTG +/+ and PTTG
-/- MEFs. MEFs were plated 18 h before treatment (timepoint 0)
and collected at the indicated timepoints for flow cytometry analysis.
Treatments included: 1, control without treatment; 2, 12-Gy
-irradiation; 3, transfection of PTTG retrovirus; 4, serum
starvation (with 0.1% FBS).
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In more detailed flow cytometry analysis, untreated WT PTTG +/+ MEFs
exhibit 6275% in G0-G1,
1527% in S, and 310% in G2-M phases,
respectively, during 96 h observation. In contrast, 3754% of
PTTG -/- MEFs were in the
G0-G1 phase, 1332% in S
phase, and 2743% in the G2-M phase (Fig. 3b
).
After
-irradiation, the number of PTTG +/+ MEFs in
G2-M phase increased to 2851%, while 4562%
of PTTG -/- MEFs were in the G2-M phase (Fig. 3b
), implying that the G2-M checkpoint is
functional in both
-irradiated PTTG +/+ and PTTG -/- MEFs (Fig. 3b
). Interestingly, introduction of PTTG into PTTG -/- MEFs via
retroviral transfection substantially increased the number of cells in
S phase (
40% at 72 h vs.
19% at baseline) while
reducing the number of cells in G2-M (
10% at
72 h vs.
29% at baseline) (Fig. 3b
), reflecting
phenotype reversal of PTTG -/- MEF cells showing a large
G2-M phase. Moreover, more than 85% PTTG +/+
MEFs were in G0-G1 after
96 h serum starvation, while only 65% PTTG -/- MEFs were in
G0-G1 with >20% PTTG
-/- MEFs still residing in G2-M after serum
starvation (Fig. 3b
).
Cytogenetic analysis of PTTG -/- MEFs showed damaged nuclei and
aberrant chromosome morphology, especially around centromeric regions.
Nuclear observation showed that 1215% PTTG -/- MEFs were
binucleated or multinucleated vs. <1% of PTTG +/+ MEFs
(Fig. 4a
). In chromosome spreads, PTTG
-/- MEFs demonstrated enhanced aneuploidy and several aberrant
chromosome morphologies (Fig. 4b
). Ten to 15% of PTTG -/- MEFs were
aneuploid vs. approximately 1% of PTTG +/+ MEFs, and
aberrant chromosome morphologies including quadriradials, triradials,
and breaks were observed in 46% of PTTG -/- metaphase spreads
examined, while no such anomalies were observed in PTTG +/+ MEFs. Also,
about 6% of PTTG -/- MEFs are apoptotic in contrast to virtually no
apoptosis observed in PTTG +/+ MEFs, as assessed by Hoescht staining.
One possibility is that the binucleated and multinucleated cells
probably contribute to the observed higher percentage of PTTG -/-
MEFs in G2-M as assessed by flow cytometry, as
well as to the aneuploidy.

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Figure 4. Abnormal Nuclear and Chromosome Morphology in PTTG
-/- MEFs
a, Binucleated and multinucleated cells in PTTG -/- MEFs. At least
1,000 cells were counted. b, Aberrant chromosome morphology in PTTG
-/- MEF metaphase cells. Three different fields are depicted, in
which quadriradial, triradial, and chromosome breaks are present as
arrowed. Aneuploidy is also apparent in these
metaphases.
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Significantly, premature centromere division was observed in
approximately 10% of chromosome spreads of PTTG -/- MEFs
vs. none in PTTG +/+ MEFs (Fig. 5
). Premature centromere division is
defined as separation of the centromere region preceding completion of
chromosome arm separation. Normally, the centromere region is the last
region to separate. In the 5 of 54 chromosome spreads of PTTG -/-
MEFs with premature centromere division, 25 chromosomes were affected
in each cell. No such premature centromere division was observed in
PTTG +/+ MEFs. However, the presence of these aberrant chromosome
structures in the PTTG -/- MEFs was not lethal for the entire cell
population.

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Figure 5. Premature Centromere Division in PTTG -/- MEFs
Four different fields are depicted, and centromere regions showing
premature division are arrowed in each field.
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DISCUSSION
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We have observed PTTG -/- mice for up to 8 months and report
here that these mice exhibit distinct cellular and physiological
phenotypes. Surprisingly, in contrast to the phenotypes observed in
yeast or Drosophila securin loss mutants, PTTG -/- mice
are viable and fertile. The nonfatal phenotype suggests that mammalian
sister chromatid separation, one of the most important transitions in
the cell cycle, is regulated by more than one mechanism involving
securin, separin, and cohesin. In a normal cell cycle, sister chromatid
pairs generated during the eukaryotic S phase remain paired through
G2 and during the initial phase of mitosis
(prophase) while chromatin is condensed and the spindle assembled
(6). Sister chromatid cohesin is ultimately disrupted at
the metaphase-anaphase transition allowing appropriate segregation
(6). Both cleavage and non-cleavage-associated
cohesin removal pathways regulate cohesin dissociation from
chromatin during prophase/prometaphase (6, 23), while
critical cohesin removal from heterochromatic regions is achieved by
separin-mediated cleavage (6). Accordingly, loss of
securin, the inhibitor to separin function, would lead to constitutive
separin activation, allowing cohesin removal from heterochromatic
regions resulting in premature centromere division. The observation of
premature centromere division in PTTG -/- MEF metaphase spreads is a
novel demonstration of the required balance between chromosomes and
interacting protein complexes. Nonetheless, the extent of premature
centromere division in most -/- cells does not appear lethal, and it
is thus likely that other mechanisms compensate for premature
centromere division in PTTG -/- MEFs. Interestingly, increased
premature centromere division and aneuploidy in aging postovulatory
mouse oocytes is coupled with reproductive failure and/or embryonic
cytogenetic aberrations (24). Thus, premature
centromere division in PTTG -/- mice may be a cause of the
observed PTTG -/- female subfertility.
Indeed, a recent observation in an in vitro
hsecurin-inactivated cell system also showed that hsecurin-deficiency
was not lethal to cells (21). These cells exhibited
defective execution of anaphase associated with incomplete sister
chromatid separation, leading to budded nuclei, chromosomal
instability, and gross aneuploidy (21). Nevertheless,
anaphase eventually did occur in most hsecurin -/- cells, suggesting
that an additional mechanism for separin regulation exists in normal
cells and basal separin proteinase activity persists in
hsecurin-deficient cells, allowing mitosis to progress. These
hsecurin-deficient cells were derived from HCT116 cells, a colorectal
cancer cell line with a stable near-diploid karyotype
(21), and the observations in these cells are similar to
those we observed in the PTTG -/- MEF cells: both cells had doubling
times comparable to their respective control cells, abnormal nuclear
morphology, chromosomal instability, and increased aneuploidy. Overall,
securin (PTTG) deficiency in either cell type did not severely affect
cell survival.
Quadriradial and triradial chromosome patterns are indications of rare
somatic chromosome exchange events. These patterns have been observed
in hereditary diseases such as Blooms syndrome and Fanconi anemia
(25, 26). Similar quadriradial and triradial
patterns have only been observed in a knockout mouse model with
targeted truncation in the murine DNA repair gene Brca2
(27). Nevertheless, the genetic mechanism underlying these
chromosomal aberrations in the PTTG knockout model may differ from that
in the Brca2 knockout model: absence of securin PTTG promotes premature
centromere division and stabilizes formation of quadriradials and
triradials; while loss of Brca2 fails to clear spontaneous chromosome
aberrations including quadriradials or triradials.
These results show that PTTG appears to be critical for maintenance of
chromosome stability and cell cycle progression, as similarly suggested
by observations in hsecurin-deficient cells (21).
Moreover, disrupted PTTG also leads to testicular and splenic
hypoplasia, thymic hyperplasia, and thrombocytopenia. Reduced testis
size has been observed in mice lacking genes required in testicular
development, including cyclin D2 (28), cdk 4
(29), cdk 4 inhibitor p19ink4d (30, 31),
E2F-1 (32), Egr 4 (33), and TATA-binding
protein-related factor Trf 2 (34). Although testis size
correlates well with mice sperm counts (31), reduced sperm
counts did not necessarily lead to infertility in these mice. Our
result implies a role for PTTG in spermatogenesis, which is under
ongoing study in our laboratory. The requirement for established
G1 and S phase regulators, including cyclin D2,
cdk 4, p19ink4d, and E2F-1, in testicular development suggests that
these proteins regulate G1 and S phase
progression during spermatogenesis. As PTTG peaks during the M phase,
PTTG could play independent and/or complementary roles to these
G1 and S phase regulators during
spermatogenesis.
Disruption of several genes, including thrombopoietin,
thrombopoietin receptor c-mpl, GATA-1, and NF-E2, resulted in
decreased megakaryocytes, decreased platelets, and increased bleeding
time (35, 36, 37). However, deletion of the
-subunit of
guanine-nucleotide-binding protein Gq resulted in increased bleeding
times with normal megakaryocyte and platelet numbers (38).
Disrupted CD39, a vascular ATP diphosphohydrolase, did not alter
megakaryocyte number, but resulted in approximately 20% lower platelet
counts and prolonged bleeding times (39). As platelet
formation from megakaryocytes involves multiple mitotic events, PTTG
could play a role during the process. Human PTTG localizes to
chromosome 5q33 (14), and 5q deletions (5q21, 5q3133)
occur in several hematological dysplasias including pediatric
thrombocytopenia and the 5q syndrome (40, 41). The human
thrombin receptor gene also localizes to 5q13 (42), and it
is therefore likely that 5q is the locus for factors required for
appropriate hematopoiesis, including PTTG.
It is likely that chromosomal and cell cycle changes caused by PTTG
loss result in unique tissue-specific phenotypic responses in
PTTG-abundant tissues, as evidenced by the observed testis hypoplasia
and thymus hyperplasia. PTTG may therefore possess cell type-specific
growth-stimulatory or -inhibitory effects. As PTTG is usually
overexpressed in tumors, it will be interesting to observe the
tissue-specific growth effect of PTTG deprivation in tumors derived
from various tissue types. Our observations demonstrate that PTTG -/-
mice exhibit unique phenotypes that will likely unravel underlying
mechanisms for PTTG action.
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MATERIALS AND METHODS
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Experimental Animals
All animal experimentation in this study was conducted in accord
with Institutional Animal Care & Use Committee (IACUC) policy and was
approved by IACUC at Cedars-Sinai Medical Center.
Plasmids and Cells
A retroviral plasmid pLPCX-PTTG was generated by subcloning
murine PTTG cDNA into pLPCX (CLONTECH Laboratories, Inc.,
Palo Alto, CA) via EcoRI and NotI sites. A viral
packaging cell line Eco293 was purchased from CLONTECH Laboratories, Inc. Retrovirus was produced by transfecting
pLPCX-PTTG into Eco293 cells and harvesting supernatants 48 h
after transfection. The viral titers were between 5 x
105/ml to 1 x
106/ml.
Generation of PTTG -/- Mice
A 16-kb NotI fragment containing the entire murine
PTTG coding region was isolated from a mouse 129 SvEv
genomic
library (Stratagene, La Jolla, CA) using a PTTG probe
(13). The targeting vector contained the equivalent of
approximately 12.5 kb murine PTTG genomic DNA with a 4-kb deletion,
including part of the first exon containing the ATG start codon, exons
2 and 3, through the middle of the third intron replaced with pGK-neo.
The targeting vector was linearized with NotI,
electroporated into J1 embryonic stem (ES) cells, and selected
in 0.4 mg/ml G418. A 345-bp fragment external to the 5'-end of the
targeting construct was used as the probe. From 800 ES colonies, 5
clones were identified with correct homologous recombination by
Southern blot analysis. A 1.7-kb hybridizing fragment corresponds to
the WT PTTG allele, while a 4.9-kb hybridizing fragment corresponds to
the targeted PTTG allele. PTTG +/- ES cells were then microinjected
into C57BL6 blastocysts, and germline transmission was observed in male
chimeras representing two separate ES cell clones. Chimeras were
crossed with the C57BL6 strain for the production of knockout mice.
Murine offspring were genotyped by either genomic Southern blot as
described above or PCR. For PCR, cycling parameters were 94 C for 20
sec, 56 C for 20 sec, and 72 C for 1 min for 30 cycles; primers
PTTG2S (5'-GGTTTCAACGCCACGAGTCG-3') and PTTG1AS
(5'-CTGGCTTTTCAGTAACGCTGTTGAC-3') were used for WT PTTG detection of
114 bp fragment; primers GENO1S (5'-GTGTGAAGGGGGAGGCTCCAATC-3') and
GENO4AS (5'-GTGCTACTTCCATTTGTCACGTCC-3') were used for targeted PTTG
detection of 596 bp fragment.
Blood samples from PTTG -/- and PTTG +/+ mice were collected for
hematological analysis including whole blood counting and blood and
bone marrow smears. Femurs were sectioned for morphological examination
and megakaryocyte counting. Bleeding time was measured as described
previously (38).
Southern and Northern Blot Analysis
For Southern blot analysis, genomic DNA from ES cells or mice
tails were digested with HindIII, electrophoresed in 1%
agarose gel, blotted onto Hybond-N membrane (Amersham Pharmacia Biotech, Arlington Heights, IL), and hybridized using QuikHyb
(Stratagene). The probe is a 345-bp fragment upstream of
exon 1 and is illustrated in Fig. 1a
.
For Northern blot analysis, total RNA was prepared using Trizol
(Life Technologies, Inc., Gaithersburg, MD),
electrophoresed in 1% formaldehyde denaturing gel, and blotted onto
Hybond-N membrane (Amersham Pharmacia Biotech Inc.,
Piscataway, NJ). A DNA fragment covering mPTTG exon 2 and 3 cDNA
sequence (372 bp) was used as probe, and glyceraldehyde-3-phosphate
dehydrogenase was used as internal control.
Cell Culture and Transfection
PTTG +/+ and -/- MEFs were prepared from embryonic day
13.5 (E13.5) embryos as described (27) and
maintained in DMEM with 10% FBS. Cells at passages 35 were plated at
4 x 105 per 60-mm dish, and either
irradiated (12 Gy) from a 137Cs Gammacell 40 irradiator (Nordion
International, Inc., Kanata, Ontario, Canada) or DMEM was added with
0.1% FBS in separate experiments. Cells were harvested at the
indicated times for cell cycle analysis. For retroviral transfection
experiments, PTTG +/+ and -/- cells were infected with PTTG
expression retrovirus produced from Eco 293 packaging cells transfected
by pLPCX-PTTG plasmid encoding full-length PTTG protein, and subjected
to cell cycle analysis.
Thymic lymphocytes were isolated from PTTG +/+ and PTTG -/- mice aged
56 wk and cultured in RPMI 1640 medium. Isolated thymocytes were also
stained for CD4 and CD8 surface expression using PE-labeled anti-CD4
(L3T4) and fluorescein isothiocyanate-labeled anti-CD8 (Ly-2) (BD
PharMingen, San Diego, CA) and analyzed using FACStar
(Becton Dickinson and Co., San Jose, CA).
Flow Cytometry
Cells were trypsinized at the indicated times, washed with PBS,
resuspended in 1 ml PBS, fixed with 2 ml cold methanol, treated with
propidium iodide and ribonuclease A, and subjected to cell cycle
analysis using FACStar (Becton Dickinson and Co.).
Nuclear and Chromosome Analysis
For nuclear analysis, MEFs grown on chamber slides were
immunostained with anti-
-tubulin and rhodamine-antigoat
secondary antibody and counterstained with Hoescht 33258
(43). For chromosome analysis, mitotic MEFs were collected
after 16 h colcemid treatment (50 ng/ml), hypotonized, and fixed
with cold Carnoys fixative. Fixed cells were processed by standard
cytogenetic procedures. Chromosome number and gross rearrangements were
determined in at least 50 metaphase cells.
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ACKNOWLEDGMENTS
|
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We are grateful to S. Ren and M. Zutler for technical help, R.
Schreck for helpful suggestions in metaphase spread preparation, and S.
Spira for megakaryocyte analysis.
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FOOTNOTES
|
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This work was supported by the Doris Factor Molecular Endocrinology
Laboratory.
Abbreviations: cdk, Cyclin-dependent kinase; ES, embryonic
stem; Gy, gray; hsecurin, human securin; MEF, mouse embryonic
fibroblast; PTTG, pituitary tumor transforming gene; Rb,
retinoblastoma; WT, wild-type.
Received for publication June 8, 2001.
Accepted for publication July 24, 2001.
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