Cytokine gene expression in splenic CD4+ and CD8+ T cell subsets of genetically resistant and susceptible chickens infected with Marek's disease virus.

T cells from the spleens of B(19)/B(19) and B(21)/B(21) chickens infected with MDV JM-16 strain were fractionated by flow cytometry at 4, 10, 21 days post infection (d.p.i.). The expression of cytokine and viral genes (meq and glycoprotein B (gB)) was measured by real-time RT-PCR. It was determined that CD4(+) and CD8(+) T cells had both become infected with Marek's disease virus (MDV) in both chicken lines. There was significantly higher expression of meq in CD4(+) T cells compared to CD8(+) T cells at 10 and 21 d.p.i. Furthermore, at 10 and 21 d.p.i., there was a tendency for higher expression of meq in both T cell subsets of B(19) chickens compared to those of B(21) chickens. There were temporal changes in the expression of cytokines, interferon (IFN)-gamma, interleukin (IL)-18, IL-6, and IL-10, in various T cell subsets. Among these changes, there was an increase in IL-10 expression in both T cell subsets at different time points, especially in the susceptible line at 10 and 21 d.p.i. Our results indicate that cytokines could be differentially induced by MDV in CD4(+) and CD8(+) T cell subsets and that IL-10 may play a role in the modulation of immune response to MDV. However, an association between cytokine gene expression in T cell subsets and resistance or susceptibility to MD was not established.

Synthesis of chicken major histocompatibility complex class II oligomers using a baculovirus expression system.

Chicken major histocompatibility complex (MHC) B21 and B19 haplotypes are associated with resistance and susceptibility to Marek's disease (MD), respectively. T-cell-mediated immune response is crucial in coordinating protection against Marek's disease virus (MDV) infection, but it has been difficult to identify and characterize antigen-specific T-cells. MHC class II tetramers and oligomers have been widely used for characterization of antigen-specific T-cells in the context of infectious and autoimmune diseases. Thus, the objective of this study was to synthesize chicken MHC class II oligomers of B21 and B19 haplotypes for the future identification of antigen-specific T-cells. To achieve this objective, full-length coding sequences of chicken MHC class II B21 and B19 molecules were amplified and the molecules were expressed as fusion proteins, carrying Fos and Jun leucine zipper (LZ), histidine-tag and biotin ligase recognition site sequences, using a baculovirus expression system. Recombinant MHC-II were loaded with self-peptides, which stabilized the heterodimer in SDS-PAGE and allowed the detection of these molecules in Western blots with a conformation-specific anti-chicken MHC class II antibody. Biotinylated MHC molecules were conjugated to streptavidin to form oligomers, which were resolved under the transmission electron microscope through immuno-gold labelling, thus confirming success of oligomerization. In conclusion, chicken MHC class II oligomers may be used in the future to study the antigen-specific CD4+ T-cell compartment.

Cloning and characterization of chicken stromal cell derived factor-1.

Stromal cell derived factor-1, SDF-1, belongs to the CXC family of chemokines and has been identified in mammals, amphibians, and fish. This chemokine has a diverse array of functions in organogenesis, hematopoeisis, B cell development and recruitment of immune system cells. Here, we report the cloning of the chicken SDF-1 ortholog and examine its temporal and spatial expression. The chicken SDF-1 cDNA contained an open reading frame encoding a predicted protein of 89 amino acids, which shared 40-75% identity to SDF-1 protein in other species. Protein folding simulation predicted a tertiary structure very similar to that obtained for human SDF-1. Recombinant chicken SDF-1 was produced using a prokaryotic expression system and the recombinant protein was shown to be biologically active in a calcium flux assay. The SDF-1 gene was found to be expressed ubiquitously and constitutively in adult tissues and was present as early as the primitive streak stage of chicken embryos.

The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events.

In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu and Cdelta flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cmu and Cdelta regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu and Cdelta regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.

Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.

Requirements for ectopic homologous recombination in mammalian somatic cells.

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.

Interchromosomal recombination is suppressed in mammalian somatic cells.

Homologous recombination occurs intrachromosomally as well as interchromosomally, both in mitotic (somatic) cells as well as meiotically in the germline. These different processes can serve very different purposes in maintaining the integrity of the organism and in enhancing diversity in the species. As shown here, comparison of the frequencies of intra- and interchromosomal recombination in meiotic and mitotic cells of both mouse and yeast argues that interchromosomal recombination is particularly low in mitotic cells of metazoan organisms. This result in turn suggests that the recombination machinery of metazoa might be organized to avoid the deleterious effects of homozygotization in somatic cells while still deriving the benefits of species diversification and of DNA repair.

High-frequency gene conversion between repeated C mu sequences integrated at the chromosomal immunoglobulin mu locus in mouse hybridoma cells.

The occurrence of mitotic recombination between repeated immunoglobulin mu gene constant (C mu) region sequences stably integrated at the haploid chromosomal immunoglobulin mu locus in murine hybridoma cells was investigated. Recombination events are detected as changes in hapten-specific immunoglobulin M production. Recombination occurs with high frequency (0.5 to 0.8%) by a mechanism consistent with gene conversion. A double-strand break repair-like mechanism is suggested by the finding that repair of a 2-bp deletion mutation and a 2-bp insertion mutation occurs with parity in a donor-directed manner. The results also suggest that the gene conversion process is directional in that the 5' C mu region sequence is preferentially converted.

Restoration of a normal level of immunoglobulin production in a hybridoma cell line following modification of the chromosomal immunoglobulin mu gene by gene replacement.

In the present investigation, we have measured the levels of IgM mRNA and protein in recombinant cell lines in which the chromosomal immunoglobulin mu gene has been modified by gene replacement (gene conversion or double reciprocal recombination) or vector integration (single reciprocal recombination) events. Our studies reveal that chromosomal immunoglobulin mu genes modified by gene replacement are expressed at wild-type levels whereas those modified by vector integration have lower levels of immunoglobulin mu gene expression. These results suggest that gene replacement may a preferred method for the construction of hybridoma and myeloma cell lines producing optimized immunoglobulins and for studies of immunoglobulin gene function.

Analysis of mutations introduced into the chromosomal immunoglobulin mu gene.

We have introduced a pSV2neo-derived vector that contains a 2-base-pair (bp) deletion in its immunoglobulin mu gene constant region into hybridoma cells bearing a single copy of the wild-type chromosomal immunoglobulin mu gene. Homologous recombination between the transferred mutant C mu region and the wild-type chromosomal C mu region is expected to introduce the 2-bp deletion into the chromosomal mu gene, generating recombinant cells synthesizing noncytolytic IgM. Analysis of the DNA in independent noncytolytic transformants indicates that in one case the mu gene has the structure expected for correct homologous recombination. Unexpectedly, the remaining transformants, bear chromosomal mu gene deletions.

Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell.