Cloning and characterization of partial cDNAs for woodchuck cytokines and CD3epsilon with applications for the detection of RNA expression in tissues by RT-PCR assay.
Immunologic reagents and methodology are essential to develop further the woodchuck and woodchuck hepatitis virus (WHV) as a model of immune response, inflammation, and immunotherapy in hepatitis B virus (HBV) infection. Partial cDNA clones for the woodchuck CD3epsilon marker of T cells (536 bp) and for selected woodchuck cytokines were developed, including IL-1beta (332 bp), IL-2 (249 bp), IL-4 (205 bp), IL-10 (476 bp), IFN-gamma (476 bp), and TNF-alpha (381 bp). This panel of markers includes sets to measure RNAs for T cells (CD3epsilon), immune response induction (IL-1beta, IL-2), TH subsets (TH1, IL-2/IFN-gamma vs. TH2, IL-4/IL-10), and effector molecules that regulate hepadnavirus replication and liver injury (IFN-gamma, TNF-alpha). Primers representing highly conserved segments of genes from other species were used to derive the partial cDNA clones. Target RNA was obtained from woodchuck peripheral blood mononuclear cells (PBMC) that were stimulated in vitro with ConA, LPS, and human rIL-2. The cDNA clones were validated by 1) comparison with other species for homologies in the nucleotide and predicted amino acid sequences and 2) a first generation assay demonstrating induction of the respective RT-PCR products in stimulated woodchuck PBMC. The corresponding RNAs were also detectable in most cases in the total RNA from the livers of uninfected and WHV-infected woodchucks and differential expression of IFN-gamma and TNF-alpha RNAs was suggested. Second generation, semi-quantitative assays for the RNAs were validated using RT-PCR and dot-blot hybridization with 32P-oligomers derived from the internal sequences of the respective clones. Continued study of the woodchuck immune response to WHV infection using these assays will provide insight into the kinetics and immune mechanisms that initiate and maintain chronic hepadnavirus infection and, hence, enable development of improved immunotherapies for established chronic HBV infection.
Nakamura
I
I
Division of Molecular Virology and Immunology, Georgetown University Medical Center, Rockville, Maryland 20852, USA.
Nupp
J T
JT
Rao
B S
BS
Buckler-White
A
A
Engle
R E
RE
Casey
J L
JL
Gerin
J L
JL
Cote
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017252:CD3 Complex / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004195:Disease Models, Animal; D006509:Hepatitis B / Q000276:immunology / Q000821:virology; D017919:Hepatitis B Virus, Woodchuck; D006801:Humans; D008099:Liver / Q000276:immunology; D008392:Marmota / Q000235:genetics* / Q000276:immunology* / Q000821:virology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
D017252:CD3 Complex; D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA