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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 168-174
Targeted Inactivation of the Coagulation Factor IX Gene Causes
Hemophilia B in Mice
By
Ramendra Krishna Kundu,
Frank Sangiorgi,
Lang-Ying Wu,
Kotoku Kurachi,
W. French Anderson,
Robert Maxson, and
Erlinda M. Gordon
From the USC Gene Therapy Laboratories, the Division of
Hematology-Oncology, the Departments of Pediatrics and Biochemistry and
Molecular Biology, Childrens Hospital Los Angeles, Los Angeles; the
University of Southern California School of Medicine, Los Angeles, CA;
and the Department of Human Genetics, University of Michigan, Ann
Arbor.
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ABSTRACT |
Hemophilia B is a leading target for gene therapy because current
therapy is not optimal. Hence, a murine model of factor IX (F. IX)
deficiency was generated to develop gene therapy strategies for
hemophilia B. A targeting vector was created by replacing a 3.2-kb
segment of the gene encompassing the catalytic domain with a
phosphoglycerokinase promoter-driven neomycin resistant (neor) gene cassette. The transfected embryonic stem cell
clones generated chimeric male mice, and germ line transmission of the
inactivated F. IX gene was observed in their offsprings. Southern
analysis confirmed the mutant genotype in hemizygous male and carrier
female mice. F. IX transcripts were not detected in liver RNA isolated from hemizygous mice, and lower levels of F. IX mRNA were noted in
carrier female mice when compared with those of normal litter mates. As
expected, the mean F. IX coagulant titer of affected male mice was 2.8 U/dL (n = 10), while the mean F. IX titer of carrier female mice was
35 U/dL (n = 14), compared with 69 U/dL (n = 9) for the normal
female mice and 92 U/dL (n = 22) for normal male and female litter
mates. Further, the tail bleeding time of hemizygous mice was markedly
prolonged (>3 hours) compared with those of normal and carrier female
litter mates (15 to 20 minutes). Seven of 19 affected male mice died of
exsanguination after tail snipping, and two affected mice died of
umbilical cord bleeding. Currently, there are 10 affected mice
surviving at 4 months of age. Aside from the factor IX defect, the
carrier female and hemizygous male mice had no liver pathology by
histologic examination, were fertile, and transmitted the F. IX gene
mutation in the expected Mendelian frequency. Taken together, we have
generated a F. IX knockout mouse for evaluation of novel gene therapy
strategies for hemophilia B.
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INTRODUCTION |
HEMOPHILIA B, AN X-LINKED clotting
disorder that affects one in 30,000 males, is caused by a deficiency of
functional clotting factor IX (F. IX) protein.1-3 The human
F. IX gene is a single copy gene residing on X-chromosome q
27.1.4 The complete sequence has been
determined5 and is at least 34 kb in size, consisting of
eight exons (a to h) and seven introns. Exon a encodes the signal
peptide, exons b and c encode the propeptide and gamma-carboxy glutamyl
domain, exons d and e encode epidermal growth factor (EGF)-like
domains, exon f encodes the activation domain where proteolytic
processing of the mature F. IX molecule occurs, and exons g and h
encode the catalytic domain of the F. IX protein. The human F. IX cDNA
is about 2.8 kb in size, and has a long 3 untranslated tail, the
function of which is still not known. The mouse F. IX cDNA sequence is
known6 and there exists a 68% sequence homology between
mouse and human F.IX protein at the amino acid level. Most of the human
mutations are found within the 2.2-kb coding region and are due to
either transitions (from purine to purine or pyrimidine to pyrimidine)
or transversion (purine to pyrimidine) at the CpG dinucleotide region
and other sites, while deletions and insertions account for only 15%
of all factor IX mutations.7-10 The catalytic region
comprises the largest domain and accounts for the largest number of F. IX mutations responsible for the moderately severe to severe forms of
hemophilia B in humans. Because hemophilia B is a prospective target
for gene therapy, we generated a F.IX knock-out mouse model for testing of gene therapy vectors and for further study of the F.IX gene function.
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MATERIALS AND METHODS |
Mouse F. IX gene isolation and construction of the targeting vector.
An 18.6-kb mouse F. IX genomic clone was isolated from a 129/Sv lambda
Fix II phage library (Stratagene, La Jolla, CA) by screening with a mouse factor IX cDNA11 and it contained
three exons corresponding to the last three exons (activation domain and catalytic domain) of the human F. IX gene. From the genomic clone,
7.0-kb Xba I, 8.0-kb Xba I, and 11.6-kb Xho
I-Not I fragments were respectively subcloned into the
pBluescript II SK vector and mapped using standard techniques. The
catalytic domain (exon g and h) was disrupted by its replacement with a
neomycin resistance gene cassette driven by a phosphoglycerol kinase
promoter (PGK-neor).12 The targeting vector was
made from the 11.6-kb lambda clone by replacement of a 3.2-kb
BamHI fragment, which contained exon g and exon h with the
1.6-kb Xho I fragment of PGK-neor. A negative
selection marker13 was created by subcloning a 2.1-kb
Xho I fragment containing the HSV-tk gene from pXhoIMC1tk (gift
from Dr Paul Hasty, M.D. Anderson Cancer Center, Houston, TX) into the Xho I site of the 11.6-kb lambda clone. Hence, the resultant vector had a 5.7-kb region of homology with the F.IX gene at
the 5 end and a 2.6-kb homology at the 3 end, a
PGK-neor cassette replacing exons g and h, with a HSV-tk
cassette at its Xho I site.
Generation of the F. IX-deficient mice.
The targeting vector was linearized by Not I digestion,
electroporated into the CCE line of embryonic stem (ES) cells (from K. Lyons, UCLA, Los Angeles, CA) (1 × 107
cells), and selected with G418 (0.4 mg/mL) and ganciclovir (2 µmol/L).14-16 The G418 resistant colonies were selected,
expanded, and screened for homologous recombination by Southern blot
technique.17 Genomic DNA from the ES cell clones was
digested with Xba I, electrophoresed on a 0.6% agarose gel,
and transferred to a nylon membrane. The membrane was hybridized with a
0.5-kb 5 external probe (Fig 1, Probe A) isolated by Xho
I digestion of the pBluescript II SK lambda 8 vector. The sizes of the
XbaI fragments of the wild-type (WT) gene and the mutant (MT)
allele were 8.0-kb and 6.4-kb, respectively (Fig 2, Probe A). An
HindIII-NotI 1.5-kb 3 probe (Fig 1, Probe B) also
confirmed the sizes of 3.9 kb for WT and 4.6 kb for the MT after
digestion of the DNA with Xba I (Fig 2, Probe B). The presence
of the single integration event was confirmed by hybridization with a
Pst I-Xba I 0.637-kb neo fragment without the
endogenous phosphoglycerol kinase promoter (data not shown). The
targeted ES cell clones were then injected into blastocysts derived
from C57BL6/J mice, and transferred into the uteri of pseudopregnant CD-1 female (USC Transgenic Core Facility). The chimeric males were
mated with C57BL6/J females, and germ-line transmission of the ES
cell-derived phenotype was determined by the presence of an agouti coat
color and further confirmed by Southern analysis of DNA. The carrier
female mice of the first (F1) generation were again mated with C57BL6/J
males to obtain males with F. IX null alleles, with the genotype again
confirmed by Southern analysis. The genotypes of the carrier female and
hemizygous male mice were determined by digestion of the tail DNA with
XbaI and hybridized with the same 5 external (Probe A) and
3 probe (Probe B) as described earlier.
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| Fig 1.
Targeted disruption of the murine F. IX gene by
homologous recombination. An 18.6-kb genomic map of the mouse F. IX
gene showing exons f, g, and h. The targeting vector was made from the
11.6-kb Xho I-Not I fragment of the lambda clone. The
targeted allele contained a neo gene inserted into exon g and exon h.
The 3.2-kb BamHI fragment covering exons g and h and the
introns was replaced by a 1.6-kb neor cassette. The 2.1-kb
thymidine kinase cassette was cloned into the Xho I site. A
0.5-kb fragment (Probe A), external to the Xho I site of the
targeting vector, and an HindIII-Not I 1.5-kb fragment (Probe B) were used to screen the ES cell clones and mouse tail DNA. A
637-bp neo gene probe was also used to screen the same clones
(data not shown). B, BamHI; E, EcoRI; H,
HindIII; N, Not I; X, Xho I; Xb, Xba I;
TK, thymidine kinase gene; neo, neomycin gene. The dotted line
represents a portion of the genomic map, which was not included in the
 clone, but was identified by subsequent mapping after Southern blot
hybridization of ES cell and mouse DNA. The * (asterisk) on the
Not I site denotes that it is not part of the clone .
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| Fig 2.
Southern analysis of the ES cell clones. (A) The ES cell
clone (D11) and 129/Sv mouse DNA were digested with Xba I and
hybridized with probe A. The wild-type clone (+/0) showed an 8.0-kb
fragment, the recombinant disrupted clone ( /0) showed a 6.4-kb band.
(B) The same DNA samples were digested with XbaI and hybridized with probe B. The wild-type clone (+/0) showed a 3.9-kb band, while the
mutant allele (/0) showed a 4.6-kb band.
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F. IX coagulant assay.
Mouse plasma coagulant F.IX titer was measured using a modification of
the kaolin partial thromboplastin time technique,18-20 with
human F.IX-deficient plasma as substrate (GK 927327P1; George King
Biomedical Co, Overland Park, KS). Blood samples were
collected by tail snipping from 4-week-old and 6-month-old mice and
mixed with 9:1 vol/vol whole blood: 3.8% sodium citrate. Reference
standard plasma (F.IX coagulant titer: 97 U/dL; Pacific Hemostasis,
Huntersville, NC) was used as the standard for
determination of the F.IX coagulant titer.
Histopathologic examination of hematoxylin-esoin stained sections from
formalin-fixed liver tissue was conducted.
Northern analysis.
Total RNA was prepared from liver tissue of 4-week-old and 6-month-old
mice, using the one-step method with RNAzol reagent (Telstar Inc,
Friendswood, TX) according to the manufacturer's instructions. Twenty-five micrograms of total RNA was electrophoresed on 0.8% agarose gel containing 6% formaldehyde, transferred to nylon
membrane (Amersham, Arlington Heights, IL) and
cross-linked to the membrane by ultraviolet (UV) light (Stratalinker,
Stratagene). The membrane was prehybridized at 65°C with Rapid-Hyb
buffer (Amersham) for 15 minutes and hybridized with a 644-bp
radiolabeled probe, which was isolated from mouse F. IX cDNA by
digestion with EcoRV and Xho I. The fragment was
similar to the 537-bp probe, which has been shown to give a better
signal than the whole cDNA.21 The mouse glyceraldehyde
3-phosphate dehydrogenase (GAPDH) probe was used as internal control.
Reverse transcription-coupled polymerase chain reaction (RT-PCR)
analysis.
Total RNA was extracted from liver tissue of 4-week-old and 6-month-old
mice using RNAzol reagent. Briefly, 5 µg of total RNA was treated
with Superscript II Rnase H-Reverse Transcriptase (GIBCO-BRL,
Gaithersburg, MD) in a 20-µL reaction volume with random
hexamers. PCR amplifications were done with Gene Amp PCR system 9600 (Perkin-Elmer, Norwalk, CT) and Taq DNA polymerase (Qiagen Inc, Santa Clarita, CA), using 2.0 µL of cDNA
solution in an incubation volume of 50 µL. PCR amplifications were
performed at 94°C for 2 minutes followed by 30 cycles at 94°C
for 1 minute, 55°C for 45 seconds, 72°C for 45 seconds, and
final extension of the PCR product at 72°C for 7 minutes. Mouse
factor IX primers were chosen from exon f
(5-GTCACTGAAAGTAGTGAA-3) and exon h antisense
(5-GACGTACCGGGAAACCTTAGT-3). The mouse 18S ribosomal primer (an internal control) was purchased from Ambion, Austin, TX. The PCR products were analyzed by loading 4 µL for
F. IX and 2 µL for 18S from 50 µL reaction volume on a 1.6%
agarose gel and visualized by ethidium bromide staining.
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RESULTS |
Targeted inactivation of the mouse F. IX gene.
A targeting vector was designed using the genomic clone of the mouse F. IX gene isolated from the 129/Sv mouse genomic library. This vector
carries a 3.2-kb deletion spanning exon g and exon h. Both the exons
and part of the introns were replaced by a 1.6-kb neomycin resistance
gene as a positive selection marker (Fig
1). Negative selection against random integration was conferred by a
herpes simplex virus thymidine kinase (HSV-tk) gene.13 ES cells were electroporated with the targeting vector and 200 double-resistant colonies were picked and screened by Southern blot
analysis. Nine positive targeted clones were identified based on the
predicted size of the targeted allele (Fig
2, Probe A and Probe B). Hybridization with the neor probe
eliminated the possibility of an additional integration event of the
targeting vector (data not shown). One of the selected clones (D11) was
then injected into C57Bl6/J blastocysts, which generated one chimeric
female and two chimeric male mice. Germline carrier females were
obtained by mating the chimeric males with C57Bl6/J females. Because
the F.IX gene is located on the X-chromosome, only the heterozygous F. IX carrier female mice with mutant alleles were obtained in the first
generation. The carrier females, which were mated with normal C57Bl6/J
male mice, transmitted the mutant F. IX allele to the male progeny
generating affected hemizygous male mice
(Fig 3, Probe A and Probe B)
(Table 1). The F. IX-deficient male mice
were again mated with normal mice and the mutant allele was transmitted
to 50% of their progenies from four different matings
(Table 2). Further, histologic examination
of the liver samples from two different litters generated from this
mating showed normal liver architecture with no liver pathology (data not shown).
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| Fig 3.
Southern analysis of tail DNA from hemizygous male and
carrier female mice. Tail DNA from normal male (+/0), affected male (/0), and carrier female (+/) mice were digested with
Xba I and hybridized with (A) probe A and (B) probe B. The
expected sizes of the bands were the same as those detected in the ES
cell clones (see Fig 2).
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F. IX transcript levels in normal, carrier, and affected mice.
To evaluate the expression of the F. IX gene in the F. IX knockout
mice, RT-PCR was done using total RNA extracted from 4-week-old carrier
female, hemizygous male, littermate control and 6-month-old normal
mice. The PCR amplified product of 713 bp from normal 4-week-old male
and female mice was comparable to the adult level F. IX PCR product
(Fig 4). The carrier female littermate
showed a reduced level of F. IX PCR product, while the affected
hemizygous male showed no detectable PCR product. Northern blot
analysis also showed the major 3.2 kb and minor 2.2 kb transcripts of
F. IX. As expected, affected male mice had no detectable F. IX mRNA, while the carrier female had reduced F. IX mRNA levels
(Fig 5A). The results were normalized
relative to GAPDH levels (Table 3).
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| Fig 4.
RT-PCR analysis of the F. IX transcripts. Total RNA was
isolated from the livers of 4-week-old carrier female, affected male, normal litter mate, and normal adult male mice. PCR amplifications were
performed for 30 cycles under the following conditions: 94°C for 1 minute, 55°C for 45 seconds, and 72°C for 45 seconds using an
F. IX exon f sense primer and an exon h antisense primer. The length of
the PCR product corresponding to the exon f and exon h portion of the
F. IX transcripts is 713-bp (lanes 1 to 7). Lanes 1 and 7, nos. 90 and
95, normal (N) males; lane 2, no. 92, normal (N) female; lanes 3 and 4, nos. 89 and 91, hemophilic (H) males; lane 5, normal 6-month-old adult
(A) male, lane 6, no. 93, carrier (C) female; lane 8, 100-bp ladder;
mouse 18S ribosomal primers were used as loading control. The PCR
fragment obtained by amplification of the 18S rRNA transcript for all
samples is 488 bp and was shown below the corresponding lanes.
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| Fig 5.
(A) Northern analysis of the F. IX transcripts. Total
liver RNA (25 µg) samples from 4-week-old carrier female, hemizygous male, normal litter mate, and normal adult male mice were loaded into
0.8% agarose-formaldehyde gel and transferred to a nylon membrane. The
membrane was hybridized with a 0.644-kb factor IX cDNA as described in
Materials and Methods. The normal sizes of the F. IX transcripts are
3.2 kb and 2.2 kb. Lanes 1 and 7, nos. 90 and 95, normal (N) males;
lane 2, no. 92, normal (N) female; lanes 3 and 4, nos. 89 and 91, hemophilic (H) males; lane 5, control 6-month-old adult (A) male; lane
6, no. 93, carrier (C) female; below, hybridization of the filter with
a GAPDH probe as internal standard. (B) Plasma F. IX coagulant titer,
expressed as U/dL, of carrier females, hemizygous male, and normal mice
are shown in corresponding lanes.
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Phenotypic analysis of F. IX-deficient mice.
In most cases, the number of pups born from each litter was normal (six
to eight pups), and the litter mates had no structural abnormalities.
Two affected male mice died 1 day after birth of umbilical cord
hemorrhage. To confirm the occurrence of F. IX gene
inactivation, tail bleeding time and plasma F. IX coagulant titers were
measured in hemizygous males, carrier females, and normal litter mates
at 1 month of age (Fig 5B). The normal littermates, normal female mice,
and carrier female mice had clotting times of 15 to 20 minutes after
tail snipping and mean plasma F. IX titers of 92, 69, and 35 U/dL,
respectively. The mean F. IX titer of carrier females was significantly
lower than those of normal female mice (P < .003). In
contrast, the affected male mice had tail clotting times of more than 3 hours and a mean plasma F. IX titer of 2.8 U/dL
(Table 4). Seven of 19 affected male mice died of excessive blood loss after tail snipping, and two affected mice
died at 2 days of age of umbilical cord bleeding. Currently, 10 affected mice are surviving at 4 months of age.
| |
DISCUSSION |
Hemophilia B is a leading target for somatic gene therapy because
current therapy is suboptimal, and this clotting disorder would be an
excellent model for gene transfer strategies requiring systemic
delivery of gene products. The clinical manifestations of hemophilia B
may be mild, moderate, or severe. Persons with severe F. IX deficiency
have plasma levels of <1 U F. IX/dL and develop frequent spontaneous
hemarthroses, which can be crippling, and are susceptible to
life-threatening hemorrhage, which would be fatal if untreated.
Patients with F. IX levels of 2 to 5 U/dL have moderately severe
hemophilia B, while patients with 5 to 30 U/dL have mild hemophilia B
and have prolonged bleeding only after surgery or severe
trauma.22 F. IX replacement therapy is the mainstay of
treatment, requiring repeated transfusions of plasma-derived and
recently recombinant F. IX preparations. Transfusion of blood-derived
F. IX products is associated with the risk of viral transmission
including human immunodeficiency virus (HIV)-1 and hepatitis viruses.
Plasma-derived, as well as recombinant F. IX preparations, are costly
and are not affordable in 80% of the world. Hence, therapy is
frequently reactive, and quality of life is impaired without sufficient
replacement therapy. In recent years, successful albeit transient gene
therapy approaches have been reported using adenoviral, retroviral
vectors, or adeno-associated viral vectors.18-20,23-31
There has been limited success in delivering the human F. IX gene with
retroviral vectors due to inefficient gene transfer,26 and
failure of repeated adenoviral vector infusions due to immune responses
against viral gene products have been reported.32 Initial
promising progress has been recently reported with recombinant
adeno-associated viral (rAAV) vectors.33
The canine hemophilia B model has long been used to test the safety and
efficacy of F. IX concentrates, and recently, of adenoviral F. IX
vectors.3,26,29 These animals are expensive to breed and
are used for the testing of F. IX therapies before a clinical trial. A
mouse model of hemophilia B would be an alternative animal model for
testing of various gene therapy strategies, as mice are inexpensive,
easy to breed, and have a much shorter gestational period than the dog.
In this study, we report the generation of a mouse model of hemophilia
B by targeted inactivation of the mouse F. IX gene. The catalytic
domain, including both exons g and h of the mouse F. IX gene, was
selected for targeted disruption because mutations in this domain
account for the largest number of cases of hemophilia B. Two other
groups have reported successful generation of F. IX knock-out mice.
Wang et al34 generated an F. IX-deficient mouse using a
similar approach by targeted inactivation of exon h of the F. IX gene.
In contrast, Lin et al35 used the plug-socket gene
targeting method to generate the hemophilia B mouse, wherein a
functional neomycin gene and a partially deleted hypoxanthine phosphoribosyl transferase minigene replaced the promoter through exon
3 of the F. IX gene. In the latter mouse model, the frequency of the
hemizygous phenotype was only 41%, which is less than the expected
frequency for affected males. In our study (Table 1), the frequency of
male offsprings with the F. IX mutation was 50%. This finding confirms
that a F. IX mutation within the catalytic domain is not
embryologically lethal.34 Moreover, we provide further
characterization of the hemophilic phenotype and additional information
relating to the fertility and postnatal survival from two generations
of hemophilic mice (Table 2), which was not described previously. The
affected male mice were not distinguishable from the carrier or WT
litter mates on the basis of size, activity, or fertility. Histologic
examination of liver sections from affected male mice showed absence of
liver pathology. Southern analysis confirmed the genotypes of the
hemizygous male and carrier female mice. F. IX transcripts were not
detected in liver RNA isolated from the hemizygous mice, while lower
levels of F. IX mRNA were noted in liver RNA from carrier female mice
compared with those of normal litter mates. Targeted disruption of the
catalytic domain of the murine F. IX gene resulted in the creation of a
murine model of hemophilia B with the affected male mice having a
phenotype of severe to moderately severe hemophilia B. Further, carrier female mice had lower F. IX titers than normal litter mates. Seven of
19 affected mice died of exsanguination after tail snipping, two
affected mice died of umbilical cord bleeding, and 10 affected mice are
alive at 4 months of age. Taken together, we confirm that targeted
disruption of the catalytic domain of the F. IX gene results in the
generation of a mouse model for severe to moderately severe hemophilia
B, which provides a valuable tool for studying the function of the F. IX gene and for developing novel gene therapy strategies for hemophilia
B.
Our studies are limited to the genotypic and phenotypic analysis of F. IX gene expression in newborn, 4-week-old, and 4-month-old knock-out
mice. Future studies will analyze F. IX gene expression with age in the
affected and carrier female mice, compared with normal litter mates.
Further, transgenic animals expressing F. IX constructs driven by
tissue-specific promoters would be mated with the F. IX-deficient mice
to test the efficiency of the gene delivery system in rescuing the
bleeding phenotype in a transgenic setting. Studies are also in
progress to test the efficacy of various retro-, adeno-, and
adeno-associated vectors bearing human F. IX constructs driven by
tissue-specific or virus-based promoter/enhancers both in vivo and ex
vivo. Finally, the murine model of severe hemophilia B could be used
for testing the safety and efficacy of new F. IX concentrates and in
studies of immunologic tolerance.
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FOOTNOTES |
Submitted September 29, 1997;
accepted March 4, 1998.
Supported in parts by the USC Center for Liver Diseases, Grant No.
DK-93-024 from the National Institute of Digestive, Diabetes, and
Kidney Diseases (awarded to E.M.G.) (Pilot/Feasibility Project #4),
Grant No. HL53713 from the National Heart Lung and Blood Institute
(awarded to K.K.), Grant No. HD22416 from the Child Health and Human
Development Institute (awarded to R.E.M.), the National Institutes of
Health, and in part by a grant from Genetic Therapy Inc/Novartis
(awarded to W.F.A.).
Address reprint requests to Erlinda M. Gordon, MD, 1441 Eastlake Ave,
MS# 73, Norris Cancer Hospital, Rm 609, Los Angeles, CA 90033.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to D.H. Zhu for technical assistance in the ES
cell cultures and to Dr F.L. Hall for helpful suggestions in writing
this manuscript.
| |
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Abstract
Introduction
Abstract