Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.     

AID is essential for immunoglobulin V gene conversion in a cultured B cell line

Following productive V gene rearrangement, the functional immunoglobulin genes in the B lymphocytes of man and mouse are subjected to two further types of genetic modification. Class-switch recombination, a region-specific but largely nonhomologous recombination process, leads to a change in constant region of the expressed antibody. Somatic hypermutation introduces multiple single nucleotide substitutions in and around the rearranged V gene segments and underpins affinity maturation. However, in chicken and rabbits (but not man or mouse), an additional mechanism, gene conversion, is a major contributor to V gene diversification. It has been demonstrated recently that both switch recombination and hypermutation are ablated in mice and humans lacking AID, a B cell-specific protein of unknown molecular activity. Here we show that disruption of AID in the DT40 chicken B cell lymphoma leads to a failure to perform immunoglobulin V gene conversion. Thus, AID is required for all three immunoglobulin gene modification programs (gene conversion, hypermutation, and switch recombination) and acts in the initiation or execution of these processes rather than in bringing the B cell to an appropriate stage of differentiation.     

Use of chicken cell line LSCC-H32 for titration of animal viruses and exogenous chicken interferon

The chicken embryo cell line LSCC-H32 was tested for the propagation and titration of several animal viruses of the families Toga-, Reo-, Rhabdo-, Herpeto-, Orthomyxo-, Paramyxo-, and Poxviridae and compared with secondary chicken embryo cells. The LSCC-H32 cells were demonstrated to be as susceptible for most of the tested viruses as were secondary chicken embryo cells. Both produced comparably sized virus plaques. The titers of Sindbis and Semliki Forest viruses in LSCC-H32 cells were 5- to 40-fold higher than in secondary chicken embryo cells or BHK-21 cells, respectively. Furthermore, exogenous chicken standard interferon was titrated in the LSCC-H32 cells, and a 50% plaque titer reduction of the challenging vesicular stomatitis virus was achieved by 0.12 IU of a standard chicken interferon preparation. Endogenous chicken interferon could not be induced by treatment of the cells with polyinosinic acid-polycytidylic acid. Due to its high plating efficiency and metabolic activities, the LSCC-H32 cell line provides a useful cell system for the titration and large-scale production of the tested animal viruses and for the titration of exogenous chicken interferon.     

Regulated expression of the chicken ovalbumin gene in a human estrogen-responsive cell line

To study the regulation of expression of the chicken ovalbumin gene by steroid hormones, the entire ovalbumin gene and its flanking sequences were cloned together with the bacterial gene for xanthine-guanine phosphoribosyltransferase in plasmid pBR322. This recombinant plasmid was linearized and used to transform an estrogen-responsive breast carcinoma cell line (MCF-7) which was shown to possess estrogen receptors and to be estrogen responsive. Transformants were selected by their ability to grow in a medium containing mycophenolic acid and xanthine. The entire ovalbumin gene was integrated into high molecular weight DNA within all transformants analyzed and it retained its original sequence organization. Ovalbumin mRNA and protein were identified from these transformant cells and they were found to be indistinguishable from the authentic counterparts. An 8- to 10-fold increase in the amount of ovalbumin mRNA was observed to be present in cells cultured in 10(-8)M estradiol. We also constructed a hybrid gene containing the 5'-flanking sequence and the first exon of the ovalbumin gene which was linked to the xanthine-guanine phosphoribosyltransferase gene such that expression of this bacterial gene would be promoted and regulated by the chicken sequences. After introduction of this hybrid gene into MCF-7 cells, we observed that the survival of the transformed cells in our selection medium was highly dependent on the presence of estradiol. Our results indicated that the chicken ovalbumin sequence was expressed properly and was regulated to some extent by estradiol in this heterologous system.     

Light chain gene conversion continues at high rate in an ALV-induced cell line.

We have analyzed immunoglobulin light chain sequences from avian leukosis virus (ALV) induced bursal and metastatic tumors and from cell lines derived from these tumors. Sequence data presented demonstrate that ALV-induced tumors and one cell line (DT40) derived therefrom continue to diversify their light chain genes outside of the bursal environment. Diversification within these tumor cells seems to occur by gene conversion events comparable with those observed in bursal B cells. Sequence analysis of spontaneously arising surface immunoglobulin negative subclones of the DT40 cell line revealed frameshifts within the rearranged light chain genes which most likely resulted from non-functional recombination events. Superimposed gene conversion events can repair these frameshifts leading to re-expression of surface immunoglobulin.     

Use of chicken cell line LSCC-H32 for titration of animal viruses and exogenous chicken interferon.

The chicken embryo cell line LSCC-H32 was tested for the propagation and titration of several animal viruses of the families Toga-, Reo-, Rhabdo-, Herpeto-, Orthomyxo-, Paramyxo-, and Poxviridae and compared with secondary chicken embryo cells. The LSCC-H32 cells were demonstrated to be as susceptible for most of the tested viruses as were secondary chicken embryo cells. Both produced comparably sized virus plaques. The titers of Sindbis and Semliki Forest viruses in LSCC-H32 cells were 5- to 40-fold higher than in secondary chicken embryo cells or BHK-21 cells, respectively. Furthermore, exogenous chicken standard interferon was titrated in the LSCC-H32 cells, and a 50% plaque titer reduction of the challenging vesicular stomatitis virus was achieved by 0.12 IU of a standard chicken interferon preparation. Endogenous chicken interferon could not be induced by treatment of the cells with polyinosinic acid-polycytidylic acid. Due to its high plating efficiency and metabolic activities, the LSCC-H32 cell line provides a useful cell system for the titration and large-scale production of the tested animal viruses and for the titration of exogenous chicken interferon.     

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

Genetic structure of a wide-spectrum chicken gene pool

The genetic structure of 65 chicken populations was studied using 29 simple sequence repeat loci. Six main clusters which corresponded to geographical origins and histories were identified: Brown Egg Layers; predominantly Broilers; native Chinese breeds or breeds with recent Asian origin; predominantly breeds of European derivation; a small cluster containing populations with no common history and populations that had breeding history with White Leghorn. Another group of populations that shared their genome with several clusters was defined as 'Multi-clusters'. Gallus gallus gallus (Multi-clusters), one of the subspecies of the Red Jungle Fowl, which was previously suggested to be one of the ancestors of the domesticated chicken, has almost no shared loci with European and White Egg layer populations. In a further sub-clustering of the populations, discrimination between all the 65 populations was possible, and relationships between each were suggested. The genetic variation between populations was found to account for about 34% of the total genetic variation, 11% of the variation being between clusters and 23% being between populations within clusters. The suggested clusters may assist in future studies of genetic aspects of the chicken gene pool.     

Genetic trends of abdominal fat content in a male broiler chicken line

Data of chickens from a broiler-breeding program were collected and used to determine the genetic trends of absolute and relative abdominal fat content. The genetic trends were estimated by the regression of trait genetic value averages on hatch-years. Genetic values from 32,485 individuals were used for regression analysis. The genetic trend estimate for absolute abdominal fat content was +0.39 g per year, indicating that abdominal fat deposition in the analyzed line, in absolute terms, tended to increase, making the existing excess fat deposition in the broilers even worse. However, the genetic trend of relative abdominal fat content was not significant, indicating that there is no increase on abdominal fat content when it is corrected for body weight.     

Recombination of exogenous interleukin 2 receptor gene flanked by immunoglobulin recombination signal sequences in a pre-B cell line and transgenic mice

We have constructed a plasmid, pLTR100, which contains human interleukin 2 receptor light (IL-2R L) chain cDNA in the inverted orientation relative to the upstream SV40 promoter. The cDNA segment is flanked by the immunoglobulin gene recombination signal sequences so that the cDNA segment can invert and the human IL-2R L chain is subsequently expressed under the control of the SV40 promoter. A murine pre-B cell line, 38B9, transfected with pLTR100 began to express the human IL-2R L chain on the cell surface. The frequency of human IL-2R L chain positive cells increased almost linearly up to 50% for 60 days of culture after transfection. Southern blot analysis and sequencing of the DNA fragments at the recombination junction confirmed that the cDNA segment was inverted in a signal sequence-dependent manner by the variable-diversity-joining recombination process. Transgenic mice bearing the recombination substrate DNA similar to pLTR100 expressed the human IL-2 L chain in the spleen, thymus, and bone marrow, but not in the other tissues examined at the detectable level. Both IgM- and CD3-positive cells expressed the human IL-2R L chain, indicating that this artificial DNA can serve as a substrate for recombination both in B- and T-cells and that another DNA segment may be necessary to confer the cell-type specificity on the substrate DNA.     

DNA damage caused by etoposide and gamma-irradiation induces gene conversion of the MHC in a mouse non-germline testis cell line

We have explored the effects of gamma-irradiation and etoposide on the gene conversion frequency between the endogenous major histocompatibility complex class II genes Abk and Ebd in a mouse testis cell line of non-germline origin with a polymerase chain reaction assay. Both gamma-rays and etoposide were shown to increase the gene conversion frequency with up to 15-fold compared to untreated cells. Etoposide, which is an agent that stabilise a cleavable complex between DNA and DNA topoisomerase II, shows an increased induction of gene conversion events with increased dose of etoposide. Cells treated with gamma-rays, which induce strand breaks, had an increased gene conversion frequency when they were subjected to low doses of irradiation, but increasing doses of irradiation did not lead to an increase of gene conversion events, which might reflect differences in the repair process depending on the extent and nature of the DNA damage. These results where DNA damage was shown to be able to induce gene conversion of endogenous genes in mouse testis cells suggests that the DNA repair system could be involved in the molecular genetic mechanism that results in gene conversion in higher eukaryotes like mammals.     

The chicken HPRT gene: a counter selectable marker for the DT40 cell line

We describe the cloning, characterisation and chromosomal mapping of the chicken hprt gene together with the construction of two counter selectable hprt-/- DT40 derived cell lines. One of these cell lines contains a stably integrated gene encoding a conditionally active cre recombinase and thus allows efficient manipulation of targeted loci by site-specific recombination. These cell lines will enhance the utility of the hyper-recombinogenic DT40 cell line as a system for the genetic analysis of cell autonomous functions in vertebrates and as a tool for mammalian chromosome engineering.     

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.     

The poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein

Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.