Molecular cloning and expression of woodchuck granulocyte-macrophage colony stimulating factor.
ABSTRACT Granulocyte-macrophage colony stimulating factor (GM-CSF) has immunoregulatory and antiviral effects, and may thus be promising for the treatment of chronic hepatitis B. Using woodchuck hepatitis virus (WHV)-infected woodchuck as an animal model to test the efficacy and safety of GM-CSF on the therapy of chronic hepatitis B, woodchuck GM-CSF will be required due to the apparent species-specific activity of GM-CSF. The cDNA of woodchuck GM-CSF was cloned using reverse transcription-polymerase chain reaction (RT-PCR) with primers deriving from highly conserved regions of GM-CSF genes from other species. The deduced amino acids, including the signal peptide, is 138 in length and its identities to human, murine, canine and bovine GM-CSFs are 63, 49, 63, and 63% respectively. The genomic DNA of woodchuck GM-CSF was also cloned by PCR. Its organization is highly homologous to that of human and murine GM-CSF genes, consisting of four exons and three introns. Cloned woodchuck GM-CSF was expressed transiently in 293T cells. The recombinant protein expressed was found to stimulate the growth and differentiation of woodchuck bone marrow cells, indicating the protein expressed by the cloned gene is functional. These results pave the way for future studies on the potential role of GM-CSF for the treatment of chronic hepatitis B by using this animal model.
- SourceAvailable from: Pei-Jer Chen[Show abstract] [Hide abstract]
ABSTRACT: Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3(+) T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.Proceedings of the National Academy of Sciences 08/2010; 107(33):14769-74. · 9.81 Impact Factor
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ABSTRACT: The eastern woodchuck, Marmota monax, represents a useful animal model to study hepatitis B virus infection in humans. However, immunological studies in this model have been impeded by a lack of basic information about the components of the immune system such as cytokines and chemokines. To clarify the role(s) of interleukin 8 (IL-8) in chronic hepatitis B and hepatocellular carcinoma (HCC) in the woodchuck model, we cloned and characterized the woodchuck IL-8 cDNA and genomic DNA. Sequence analysis revealed that the organization of the wk-IL-8 gene is similar to that of the human IL-8 gene and consists of four exons and three introns. Woodchuck IL-8 protein exhibits the conserved ELRCXC motif of IL-8 and shows 87, 82, 82 and 79% similarity with rabbit, ovine, bovine and human IL-8 proteins, respectively. The biological activity of wk-IL-8 was demonstrated using neutrophil chemotaxis assays. Wk-IL-8 could be readily detected in both tumor and non-tumor tissues with higher expression in the non-tumor tissues in most cases. The results from this study will facilitate the investigation of IL-8 in the immunopathogenesis of hepadnavirus-related diseases by the woodchuck model.Genes and immunity 11/2008; 10(1):27-36. · 4.22 Impact Factor
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ABSTRACT: We aimed to evaluate the transcriptional characteristics of viral infection-induced woodchuck hepatocellular carcinoma (HCC), to compare the use of human, rat and mouse gene arrays for cross-species hybridization, and to look into gene expression profiles in woodchuck HCC by the combined use of these arrays. Commercially available human, rat and mouse oligonucleotide microarrays were used to determine the gene expression profiles on the same woodchuck liver samples. Differentially expressed genes between HCC and the surrounding hepatic tissues found in the arrays were selected for quantitative reverse transcription polymerase chain reaction. Despite the difference in the number of the probes from each array, the percentage of genes that were detectable was similar. Stringent microarray data analysis using both supervised and unsupervised methods identified 281 differentially expressed genes via the human array with a false discovery rate (FDR) of 0.99%, 107 genes via the rat array with an FDR of 1.85% and 78 genes via the mouse array with an FDR of 7.41%. Eleven genes were differentially changed in all three arrays that include the upregulation of NPM1, H2AFZ, EEF1G, HNRPAB, RPS18, EIF5, CKS2, ARIH1, RPS12 and RPS10, and the downregulation of EGR1. The quantitative reverse transcription polymerase chain reaction with woodchuck-specific primers confirmed the reliability of the microarray results. This study further demonstrated the utility of cross-species hybridization of microarrays on woodchuck HCC. A combined use of three types of arrays identified more differential genes in HCC than individual arrays with the human array providing the richest information among the three arrays used.Journal of Gastroenterology and Hepatology 11/2008; 24(4):605-17. · 3.33 Impact Factor
Molecular cloning and expression of gerbil
granulocyte/macrophage colony-stimulating factor
Denis Gaucher, Kris Chadee*
Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, QC, Canada H9X 3V9
Received 27 March 2002; received in revised form 28 May 2002; accepted 11 June 2002
Received by V. Larionov
Using a combination of cross species reverse transcriptase–polymerase chain reaction and30rapid amplification of cDNA ends techniques,
we cloned the cDNA encoding gerbil granulocyte/macrophage colony-stimulating factor (GM-CSF). The open reading frame had 81%
nucleotide identity with its mouse counterpart, while the mature protein had 80% homology with mature mouse GM-CSF. COS-7 cells
transfected with gerbil GM-CSF cDNA secreted high levels of bioactive GM-CSF, as their supernatant stimulated gerbil bone-marrow cell
proliferation and colony formation in semi-solid medium. q 2002 Elsevier Science B.V. All rights reserved.
Keywords: Bioassay; Cytokine; Hematopoietic factor; Granulocyte macrophage colony stimulating factor; cDNA sequence
The Mongolian gerbil (Meriones unguiculatus) is permis-
sive to several infections, and is an exciting animal model
alternative for the study of diseases that cannot infect mice
and rats. It is currently being used in studies on infectious
diseases of viral (Nakamura et al., 1999), bacterial (Blanot
et al., 1997; Hirayama et al., 1999) and parasitic (Chadee
and Meerovitch, 1984; Dubey and Lindsay, 2000; Sato and
Kamiya, 2001) origins. However, the lack of commercially
hampered immunopathological studies on those diseases.
Cross-species bioassays can be used for a few well-
conserved cytokines (interleukin (IL)-2, IL-4, tumor necro-
sis factor-a) (Campbell and Chadee, 1997), but most cyto-
kines are species-specific and inactive on cells from other
species. Furthermore, in vivo injection of cytokines from
other species is not advisable since the host could eventually
raise an immune response against the foreign molecule,
which would abrogate its effect. Thus, there is a need to
identify gerbil cytokine coding sequences and use them to
produce immunological reagents.
GM-CSF isa hematopoietic cytokine produced by several
cell types including T cells, macrophages, mast cells,
endothelial cells, epithelial cells and fibroblasts. Its main
activity is to support the production of granulocytes (mainly
neutrophils and eosinophils) and macrophages from bone
marrow progenitor cells (Metcalf, 1986). Therefore, GM-
CSF is an obvious molecule to be used clinically to counter-
act the effects of treatments that deplete those cell types
(Fox, 1994). GM-CSF is being used, among other things,
chemotherapy (Beveridge et al., 1997), in AIDS patients
during therapy (Frumkin, 1997), and is administered after
bone marrow transplantation (Weinthal, 1996).
GM-CSF also activates mature eosinophils and neutro-
phils, enhances their survival (Lopez et al., 1986), and
increases their ability to kill infectious agents (Lopez et
al., 1983). Furthermore, it enhances the killing capacity of
macrophages (Handman and Burgess, 1979), as well as their
antigen-presentation ability (Fischer et al., 1988) by increas-
ing their expression of Class II MHC, adhesion molecules
and costimulatory factors. GM-CSF is also involved in the
development (Caux et al., 1992) and activation (Salgado et
al., 1999) of dendritic cells, which are central in the induc-
tion of primary immune responses.
Because of its differentiation and activation properties on
antigen-presenting cells as well as some other immunomo-
dulatory effects, GM-CSF is an attractive molecule to be
used as an adjuvant in vaccination strategies, and several
studies have already been published in this field (Warren
Gene 294 (2002) 233–238
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.
Abbreviations: aa, amino acid(s); cDNA, complementary DNA; FBS,
fetal bovine serum; GM-CSF, granulocyte/macrophage colony-stimulating
factor; LPS, lipopolysaccharide; nt, nucleotide(s); ORF, open reading
frame; RACE, rapid amplification of cDNA ends; RT–PCR, reverse tran-
scription–polymerase chain reaction; UTR, untranslated region
* Corresponding author. Tel.: 11-514-398-7721; fax: 11-514-398-7857.
E-mail address: firstname.lastname@example.org (K. Chadee).
and Weiner, 2000). The cytokine, either injected as a solu-
ble protein, encapsulated in liposomes, or as DNA (GM-
CSF-encoding sequence), generally improved the immune
response to the co-administered antigen, protection against
disease, and this with virtually no side effects. Herein, we
report the cloning, expression and characterization of gerbil
2. Materials and methods
2.1. Collection of peritoneal cells and isolation of RNA
Resident peritoneal cells from 50–60-days-old female
gerbils (Charles River. St. Constant, Canada) were
harvested by lavage of the peritoneal cavity as described
(Mishell and Shiigi, 1980), with 10 ml of ice-cold 10-
RPMI, made of RPMI 1640 medium (Gibco, Burlington,
Canada) supplemented with 100 U/ml penicillin, 100 mg/
ml streptomycin sulfate, 24 mM HEPES (Sigma, St. Louis,
MO) and 10% FBS (Gibco). The erythrocytes were lysed
with Gey’s salts (Mishell and Shiigi, 1980), and the remain-
ing cells were resuspended in the above medium at a density
of 5 £ 106/ml. The suspension was aliquoted in a 24-well
plate at 1.5 ml/well, and the cells were incubated for 24 h at
37 8C in a humidified 5% CO2atmosphere, with 1 mg/ml
LPS (Escherichia coli 0111:B4; Sigma). The stimulated
cells were harvested and their RNA isolated with TRIzol
reagent (Gibco), according to the manufacturer’s instruc-
cDNA was generated from 2 mg total RNA in a 50-ml
reaction containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl,
3 mM MgCl2, 4 mM dithiothreitol, 1 mM dNTP (Gibco), 8
U of RNasin ribonuclease inhibitor (Promega Corp., Madi-
son, WI), 100 U of Moloney MuLV reverse transcriptase
(Gibco) and 0.2 mg oligo(dT) primer (Gibco) for regular
PCR, or 20 pmol anchor primer 50-GGC CAC GCG TCG
ACT AGT AC(T)17-30for 30-RACE. The reaction was incu-
bated at 37 8C for 1 h, and the enzyme was then inactivated
at 95 8C for 5 min. All subsequent PCRs contained 20 mM
Tris–HCl (pH 8.4), 50 mM KCl, 1.5–3.5 mM MgCl2, 40 mM
dNTP, 50 pmol of both sense and antisense primers, 2.5 U
Taq DNA polymerase and 5 ml of cDNA, in a total volume
of 50 ml. The PCR conditions always consisted of: 30 s at 95
8C, 1 min at 55 8C, and 2 min at 72 8C for 35 cycles, and a
final cycle with the extension step at 72 8C for 8 min. PCR
products were separated by electrophoresis in 1–2% agarose
gels containing ethidium bromide. All relevant DNA bands
were sliced out, purified using the Sephaglas BandPrep Kit
(Amersham Pharmacia Biotech, Baie d’Urfe ´, Canada),
inserted in the cloning vector pGEM-T Easy (Promega),
2.2. Cloning of gerbil GM-CSF
Cloning of gerbil GM-CSF cDNA was done in two steps.
First, PCR was done with the primers ‘GM-CSF_50sense’:
50-AAG GCT AAG GTC CTG AGG AGG-30, which was
based on the 50untranslated region of the mouse GM-CSF
cDNA (GenBank accession number X03019), and ‘GM-
CSFint antisense’: 50-CTT CAG GCG GGT CTG CAC
AC-30, also based on the mouse sequence. The 255 bp
product was cloned, and its sequence used to design the
primer ‘jGM-CSF sense2’: 50-TCA AAG AAG CTC TGA
GCC-30. This primer was then used in 30-RACE together
with the adapter primer 50-GGC CAC GCG TCG ACT AGT
AC-30. The reaction gave a product of 503 bp, which was
cloned and sequenced, and found to contain the rest of the
coding sequence and the complete 30UTR, up to the poly(A)
2.3. Expression of gerbil GM-CSF in COS-7 cells
DNA encoding the full gerbil GM-CSF protein was
amplified by PCR from cDNA derived from LPS-stimulated
peritoneal cells, using the primers ‘jGM-CSFXhoI sense’:
50-GCA CTC GAG GTC CTG AGG AGG ATG TG-30and
‘jGM-CSFSalI antisense’: 50-TCA GTC GAC TCA CTC
TTG GAC TGG CTC-30and Pwo DNA polymerase (Boeh-
ringer Mannheim, Laval, Canada), according to the manu-
facturer’s instructions. The product was digested and ligated
into XhoI/SalI-treated pCI-neo (Promega), to yield the plas-
mid pjGM-CSF. The insert in this plasmid was sequenced to
verify that no mistakes were introduced during the PCR
COS-7 cells were plated at a density of 4 £ 105=ml in 60-
mm culture dishes (Sarstedt, Newton, NC) in DMEM
(Gibco) supplemented with 100 U/ml penicillin, 100 mg/
ml streptomycin sulfate, 24 mM HEPES and 10% FBS,
and incubated overnight. They were then transfected with
1 mg of CsCl gradient centrifugation-purified pjGM-CSF or
pCI-neo (control vector), using LipofectAmine reagent
(Gibco) in serum-free, antibiotic-free DMEM. After a 5-h
incubation period in the presence of the DNA–liposome
complexes, the medium was removed and replaced with
DMEM containing antibiotics but no serum. The cultures
were incubated for another 72 h, and the cells and their
supernatants were harvested and kept at 4 8C.
2.4. Bone marrow cell proliferation assay
Tibias and femurs from a gerbil were flushed with cold
RPMI supplemented with 2% FBS, and the bone marrow
was collected in a 50-ml centrifuge tube. The clumps were
separated by vigorous pipetting until a single-cell suspen-
sion was obtained. The red blood cells were lysed with
Gey’s salts, and the remaining cells were resuspended at a
density of 5 £ 105=ml in 15-RPMI (RPMI 1 15% FBS) and
0.1 ng/ml LPS. The cells were distributed in a 96-well
culture plate (100 ml/well), and an equal volume of 1:10
serially diluted COS-7 supernatant containing gerbil GM-
CSF was added to the cells. Control wells received 1:10
serially diluted supernatant from COS-7 cells transfected
with the empty vector pCI-neo for 72 h. Each condition
D. Gaucher, K. Chadee / Gene 294 (2002) 233–238
was done in triplicate. The cells were incubated for 4 days at
37 8C in a humidified 5% CO2atmosphere, and 1 mCi
[3H]thymidine in 15-RPMI was added to each well in a
volume of 25 ml. The cultures were incubated for another
24 h and harvested onto glass-fiber filters, using an auto-
mated cell harvester (Skatron, Lier, Norway). The radioac-
tivity incorporated in the cells was finally measured by
scintillation counting (LKB Wallac; Pharmacia).
2.5. Colony formation assay
Bone marrow cells were obtained as described above, and
added to a density of 7:5 £ 104=ml to warm 15-RPMI (42 8C)
containing 0.1 ng/ml LPS and 0.4% Bacto Agar (Difco,
Detroit, MI). One-milliliter aliquots were then quickly
distributed in six-well plates already containing 150 ml of
undiluted COS-7 supernatant containing gerbil GM-CSF or
control supernatant. The medium was left tosolidify atroom
temperature for 15 min, and the cultures were then incu-
bated at 37 8C in a well-humidified, 5% CO2atmosphere, for
3.1. Nucleotide and amino acid sequences of gerbil GM-
A 590 bp cDNA sequence for gerbil GM-CSF was deter-
mined (Fig. 1), which starts with the initiation codon, and
includes a 438 bp ORF, as well as a complete 30-UTR
containing a polyadenylation signal sequence AATAAA
and a poly(A) tail. The ORF is 81% identical to that of
mouse GM-CSF, and encodes a 146 aa precursor polypep-
tide with an N-terminal 17 aa secretion signal peptide, based
on the mouse (Sparrow et al., 1985) and human (Wong et
al., 1985) GM-CSF proteins. The calculated molecular
weight of precursor gerbil GM-CSF is 16.9 kDa, while
that of the mature cytokine is 14.8 kDa. Mature gerbil
D. Gaucher, K. Chadee / Gene 294 (2002) 233–238
Fig. 1. Nucleotide sequence and corresponding amino acid sequence of gerbil GM-CSF cDNA. The nucleotide positions are numbered above the DNA
sequences. An asterisk identifies the stop codon and the consensus poly(A) addition sequence is shown in bold characters. The arrow shows the putative signal
peptide cleavage site and the potential N-linked glycosylation site is underlined. The GenBank accession number for gerbil GM-CSF is AF387363.
GM-CSF (Fig. 2) is 80% homologous to its mouse counter-
part. The four cysteine residues thought to form two intra-
molecular disulfide bonds in mouse, human (Schrimsher et
al., 1987) and all other known GM-CSFs are also conserved
in the gerbil molecule. While mouse GM-CSF has two
potential N-linked glycosylation sites, the gerbil protein
only has a single one (Fig. 1).
3.2. Expression and analysis of gerbil GM-CSF
COS-7 cells were transfected with the plasmid pjGM-
CSF (or pCI-neo as control) for 72 h, and their supernatants
were harvested. To determine whether the conditioned
media contained GM-CSF that was bioactive, we first
performed a bone marrow cell proliferation assay. As
shown in Fig. 3, the supernatant from the cells transfected
with pjGM-CSF induced the proliferation of gerbil bone
marrow precursor cells in a dose-dependent manner. The
highest level of proliferation was obtained with dilutions
of 5 £ 1021and 5 £ 1022, and activity was completely
titrated out at 5 £ 1026. Control supernatants did not stimu-
late appreciable cell proliferation.
The bioactivity of the supernatants was also tested in a
colony-formation assay (Fig. 4). While control supernatants
did not stimulate colony formation at all when incubated
with gerbil bone marrow precursor cells, culture medium
conditioned by COS-7 cells expressing gerbil GM-CSF
stimulated the formation of colonies composed of small,
tightly grouped cells, as well as bigger cells migrating
away from the focus of proliferation.
Concurrent experiments being carried out in our labora-
tory prompted us to clone the cDNA encoding GM-CSF in
gerbils. Using cross-species RT–PCR and 30-RACE techni-
D. Gaucher, K. Chadee / Gene 294 (2002) 233–238
Fig. 2. Alignment of the deduced amino acid sequence of mature gerbil, mouse (GenBank accession number X03019), rat (P48750), horse (AAK72108),
guinea pig (Q60481) and human GM-CSF (M10663). The amino acid residues are numbered above the alignments. Conserved residues are boxed in black,
identical residues in dark gray and similar residues in light gray. Asterisks identify the cysteine residues.
Fig. 3. Stimulation of gerbil bone marrow cell proliferation by gerbil GM-
CSF. Bone marrow cells were incubated with 1:10 serially diluted super-
natants from a 72-h culture of COS-7 cells transfected with pjGM-CSF (B)
orwiththe emptyvectorpCI-neo(O).Incorporated [3H]thymidinewasthen
determined as an indication of cell proliferation. Results are expressed as
mean cpm of triplicate cultures ^ SE.
ques, we identified a 438 bp ORF that had an 81% identity
with that of mouse GM-CSF. Gough et al. (1985) reported
that the mRNA encoding murine GM-CSF contains two
functional start codons. They suggested, on the basis of
N-terminal amino acid composition, that translation
products initiated from the first (50proximal) AUG could
be integral membrane proteins, while those starting from the
second AUG are secreted. Since the ‘GM-CSF_50sense’
primer used to clone the 50-end of the gerbil GM-CSF
coding sequence was based on the sequence immediately
upstream of the second AUG of the mouse GM-CSF
sequence, the gerbil sequence we identified and expressed
corresponds to the secreted form of the cytokine.
Mature gerbil GM-CSF protein has a homology of 80%
with its mouse counterpart. Although both molecules differ
substantially in the region 27–40, where the gerbil protein
has four extra amino acid residues, gerbil GM-CSF is still
bioactive on mouse bone marrow cells (data not shown).
This would suggest that this region is not critical for binding
to the murine receptor and for bioactivity, although it has
been previously reported that amino acid residues 24–37 of
mouse GM-CSF are critical for function (Shanafelt and
Kastelein, 1989). Furthermore, it was also reported that
residues Asp-92, Thr-98 and Asp-102 of mouse GM-CSF
are essential for proper interaction of the ligand with its
receptor (Shanafelt et al., 1991). In gerbil GM-CSF, while
the last two of those residues are conserved (Thr-102 and
Asp-106), the first one is not. However, its corresponding
residue, Glu-96, is similar in shape and function to Asp, and
therefore could act the same way. Alternatively, the preced-
ing residue, Asp-97, could be the one interacting with the
receptor. COS-7 cells transfected with a plasmid encoding
gerbil precursor GM-CSF secreted high levels of bioactive
GM-CSF. Supernatants from those cells stimulated cell
proliferation from gerbil bone marrow precursors and
formation of colonies in semi-solid medium that appeared
exactly as previously described GM-CSF- stimulated colo-
nies (Metcalf, 1985).
The cloning, characterization and expression of gerbil
GM-CSF can now allow the generation of gerbil-specific
immunological reagents, such as recombinant GM-CSF,
anti-GM-CSF antibodies, molecular probes and GM-CSF-
encoding expression vectors. Clearly, these reagents will be
useful in the study of the role of GM-CSF in different patho-
logical conditions, or in vaccination or gene therapy trials.
This study was supported by a grant from the Natural
Sciences and Engineering Research Council of Canada.
Research at the Institute of Parasitology is partially funded
by the Fonds pour la Formation de Chercheurs et l’Aide a ` la
recherche du Que ´bec. D.G. is the recipient of a McGill
University Graduate Fellowship.
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