Analysis of the DNA sequencing quality and efficiency of the Apollo100 robotic microcycler in a core facility setting.

Sanger, or dideoxynucleotide sequencing, is an important tool for biomolecular research. An important trend in DNA sequencing is to find new and innovative ways to provide high-quality, reliable sequences in a more efficient manner, using automated capillary electrophoresis. The Apollo100 combines Sanger cycle sequencing and solid-phase reversible immobilization for product purification in a single instrument with robotic liquid handling and microfluidic (Microscale On-chip Valve) chips that have onboard thermal cycling and pneumatic mixing. Experiments were performed to determine how the DNA sequencing results from the Apollo100 compared with conventional, manual methods used in a core facility setting. Through rigorous experimentation of multiple baseline runs and a dilution series of template concentration, the Apollo100 generated sequencing that exceeded 900 bases with a quality score of 20 or above. When comparing actual client samples of amplicons, plasmids, and cosmids, Apollo100 sequencing results did not differ significantly from those reactions prepared manually. In addition, bacterial genomic DNA was sequenced successfully, directly with the Apollo100, although results were of lower quality than the standard manual method. As a result of the microscale capabilities, the Apollo100 offers valuable savings with respect to the quantity of reagents consumed compared with current manual sequencing methods, thereby continuing the demand for smaller template and reagent requirements. In conclusion, the Apollo100 can generate high-quality DNA sequences for common templates equivalent to those produced using manual sequencing methods and increases efficiency through reduced labor and reagents.

Development of a toxicological gene array and quantitative assessment of this technology.

High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to beta-naphthoflavone (beta-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after beta-NF administration. Significant upregulation of Cyp1a2 was noted at beta-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15- and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18-60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability.

Gene-targeted B-deficient mice reveal a critical role for B cells in the CD4 T cell response.

The role of B cells in promoting T cell responses is still controversial. In this study, we use JHD mice which have a targeted mutation in the JH gene and are thus rendered deficient in B cells to address this issue. We show here that immunization of JHD mice with soluble antigen fails to prime CD4 T cells, for either clonal expansion or delivery of immunological help for antibody responses. This lack of CD4 T cell priming in JHD mice corresponds to a 3- to 9-fold lower co-stimulatory activity of antigen-presenting cells (APC) from the JHD mice, as measured by anti-CD3-induced proliferative responses of CD4 T cells. This in turn is due to a defect of APC from JHD mice in response to T cell-mediated induction of co-stimulatory activity. As the development of macrophages and dendritic cells is unaffected in the JHD mice, our results demonstrate that B cells play a critical role in CD4 T cell priming, possibly by delivering a critical co-stimulatory activity for clonal expansion of CD4 T cells.

Light chain editing in kappa-deficient animals: a potential mechanism of B cell tolerance.

The genetic organization of the kappa and lambda light chain loci permits multiple, successive rearrangement attempts at each allele. Multiple rearrangements allow autoreactive B cells to escape clonal deletion by editing their surface receptors. Editing may also facilitate efficient B cell production by salvaging cells with nonproductive light chain (L chain) rearrangements. To study receptor editing of kappa L chains, we have characterized B cells from mice hemizygous for the targeted inactivation of kappa (JCkD/wt) which have an anti-DNA heavy chain transgene, 3H9. Hybridomas from JCkD/wt mice exhibited an increased frequency of rearrangements to downstream Jk segments (such as Jk5) compared with most surveys from normal mice, consistent with receptor editing by sequential kappa locus rearrangements in JCkD/wt. We observed an even higher frequency of rearrangements to Jk5 in 3H9 JCkD/wt animals compared with nontransgenic JCkD/wt, consistent with editing of autoreactive kappa in 3H9 JCkD/wt. We also recovered a large number of 3H9 JCkD/wt lines with Vk12/13-Jk5 rearrangements and could demonstrate by PCR and Southern analysis that up to three quarters of these lines underwent multiple kappa rearrangements. To investigate editing at the lambda locus, we used homozygous kappa-deficient animals (JCkD/JCkD and 3H9 JCkD/JCkD). The frequencies of V lambda 1 and V lambda 2 rearrangements among splenic hybridomas in 3H9 JCkD/JCkD were reduced by 75% whereas V lambda X was increased 5-10-fold, compared with nontransgenic JCkD/JCkD animals. This indicates that V lambda 1 and V lambda 2 are negatively regulated in 3H9 JCkD/JCkD, consistent with earlier studies that showed that the 3H9 heavy chain, in combination with lambda 1 binds DNA. As successive lambda rearrangements to V lambda X do not inactivate V lambda 1, the consequence of lambda editing in 3H9 JCkD/JCkD would be failed allelic exclusion at lambda. However, analysis of 18 3H9 JCkD/JCkD hybridomas with V lambda 1 and V lambda X DNA rearrangements revealed that most of these lines do not have productive lambda 1 rearrangements. In sum, both kappa and lambda loci undergo editing to recover from nonproductive rearrangement, but only kappa locus editing appears to play a substantial role in rescuing autoreactive B cells from deletion.

Antigen-specific human antibodies from mice comprising four distinct genetic modifications.

Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus.

B lymphocyte differentiation is characterized by an ordered series of Ig gene assembly and expression events. In the majority of normal B cells, assembly and expression of Ig heavy (H) chain genes precedes that of light (L) chain genes. To determine the role of the Ig heavy chain protein in B cell development and L chain gene rearrangement, we have generated mice that cannot assemble Ig H chain genes as a result of targeted deletion of the JH gene segments in embryonic stem cells. Mice homozygous for this deletion are devoid of slg+ B cells in the bone marrow and periphery. B cell differentiation in these mice is blocked at the large, CD43+ precursor stage. However, these precursor B cells do assemble kappa L chain genes at a low level in the absence of mu H chain proteins. These data demonstrate that rearrangement and expression of the mu H chain gene is not absolutely required for kappa L chain gene rearrangement in vivo. Expression of mu chains may facilitate either efficient L chain gene rearrangement or the survival of cells that have rearranged light chain genes by promoting the differentiation of large, CD43+ to small, CD43- pre-B cells.

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.

Multicolor fluorescence in situ hybridization and pulsed field electrophoresis dissect CMT1B gene region.

We have used multicolor fluorescence in situ hybridization of banded chromosomes to orient Fc gamma RII and clone 1054 on a single early metaphase chromosome band (1q22) representing about 2% of the physical map of chromosome 1 in the Charcot-Marie-Tooth (CMT1B) gene region. These two cloned fragments are on the same partially digested 900-kb MluI fragment detected by pulsed field gel electrophoresis. When applied to data from an earlier study, multicolor in situ hybridization results further refined the CMT1B genetic location from an 18 cM interval to a 6 cM interval and the physical map from 15% of chromosome 1 to 3% of chromosome 1. Occasionally the three Fc gamma RII immunoglobulin receptor genes within the 200-kb region are resolved in individual metaphase chromatids.

Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI.

Complementary DNA clones encoding mouse cytokine synthesis inhibitory factor (CSIF; interleukin-10), which inhibits cytokine synthesis by TH1 helper T cells, were isolated and expressed. The predicted protein sequence shows extensive homology with an uncharacterized open reading frame, BCRFI, in the Epstein-Barr virus genome, suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host.

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1.

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.

Reactivity of cloned, expressed human Fc gamma RIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of Fc gamma RIII.

We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N-glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil-derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies.

Cloned and expressed human Fc receptor for IgG mediates anti-CD3-dependent lymphoproliferation.

We have utilized gene transfer experiments to investigate the role of a human monocyte receptor for IgG (Fc gamma RII) in mouse IgG1 anti-CD3 (Leu 4)-induced lymphoproliferation in vitro. Mouse Ltk- cells expressing human Fc gamma RII or a mutant of Fc gamma RII lacking the entire cytoplasmic domain of the receptor mediate anti-CD3-induced lymphoproliferation in cultures of adherent cell-depleted human PBMC. Expression of an Fc gamma RII mutant lacking transmembrane and cytoplasmic domains (soluble Fc gamma RII) in COS7 cells yielded a secreted receptor which retained affinity for IgG, even in the absence of the mutant receptor's N-linked oligosaccharides. Soluble Fc gamma RII inhibits rosette formation by human IgG-sensitized RBC and the Fc gamma RII-bearing cell line K562, but does not sitmulate anti-CD3-induced lymphoproliferation under the conditions tested.

Isolation and expression of cDNA clones encoding a human receptor for IgG (Fc gamma RII).

We have cloned and expressed a cDNA encoding a human receptor for IgG (Fc gamma R) from the monocyte cell line U937. The deduced structure is a 35-kD transmembrane protein with homology to the mouse Fc[gamma 2b/gamma 1] receptor amino acid sequence of approximately 60% in the extracellular domain. The signal sequence is homologous to the mouse Fc gamma R alpha cDNA clone, while the transmembrane domain shares homology with mouse Fc gamma R beta cDNAs. The cytoplasmic domain is apparently unique. The extracellular domain shows significant homology to proteins of the Ig gene superfamily, including the human c-fms protooncogene/CSF-1 receptor. Mouse Ltk- cells transfected with the human Fc gamma R cDNA express a cell-surface receptor that selectively binds human IgG and is recognized by the anti-Fc gamma RII mAb IV.3. Antibodies against peptides derived from the human Fc gamma R sequence specifically stain U937 cells, but not an Fc gamma RII-bearing B-lymphoblastoid cell line (Daudi). These results identify the human Fc gamma RII as the homologue of mouse Fc[gamma 2b/gamma 1] R, and provide evidence for heterogeneity of Fc gamma RII expressed on monocytes and B cells.

Potentiating and suppressive IgE-binding factors are expressed by a single cloned gene.

We have investigated expression of an IgE-binding factor (IgE-BF) cDNA in both COS-7 monkey kidney cells and Chinese hamster ovary cells. Transient expression of the IgE-BF clone in either cell type yielded IgE-BF, which potentiated an in vitro IgE response and had an affinity for lentil lectin. In contrast, when the transient expression experiments were carried out in the presence of tunicamycin, the factors no longer bound to lentil lectin. Moreover, IgE-BF expressed under these conditions suppressed an in vitro IgE response. IgE-BF lacking affinity for lentil lectin and suppressing the IgE response also resulted from transient expression of the IgE-BF gene in the presence of glycosylation inhibiting factor, a phospholipase inhibitory protein. Thus, IgE-BF that either potentiate or suppress the IgE response can be expressed from a single cloned gene; the difference in biological activities appears to be determined principally by the type of glycosylation of the common polypeptide chain. Previous work showed that IgE-BF bears an antigenic determinant recognized by the anti-Ia monoclonal antibody OX3. IgE-BF produced in the presence of tunicamycin, and IgE-BF expressed from a mutant cDNA lacking one of two carbohydrate-attachment sites, lacked the OX3 determinant. Thus, the OX3 determinant on IgE-BF appears to be associated with a site of N-linked glycosylation.

cDNA clones encoding murine IgE-binding factors represent multiple structural variants of intracisternal A-particle genes.

Previously [Moore, K. W., Jardieu, P., Mietz, J. A., Trounstine, M. L., Kuff, E. L., Ishizaka, K. & Martens, C. L. (1986) J. Immunol. 136, 4283-4290], we examined a T-hybridoma-derived cDNA clone, 8.3, that encodes a biologically active murine IgE-binding factor (IgE-BF), and we showed that it was a variant member of the endogenous retroviral gene family related to mouse intracisternal A particles (IAPs). We have now characterized four more IgE-BF cDNA clones by heteroduplex and restriction enzyme analysis and found that they all represent different structural variants of the full-size IAP genomic element. In clones 8.3 and 10.2, which have been fully sequenced, the open reading frames span deletions 3.4 and 1.9 kilobases (kb) long, respectively, and specify different gag-pol fusion polypeptides. Clone 9.5 contains a 2.1-kb deletion entirely within the pol region. Two other clones (4.2 and 11.7) contain no internal deletion and may represent truncated cDNA copies of full-size (7.2 kb) IAP gene transcripts. Structural variants very similar to clone 10.2 are common in the mouse genome, and clone 9.5 is also probably not a unique gene form. The sequences of clones 8.3 and 10.2 are different in detail, but each is closely homologous to a randomly cloned mouse genomic IAP element throughout the gag-related portions of their open reading frames. Antibodies against two oligopeptides specified by the sequence of clone 8.3 immunoprecipitated IAP-related proteins from mouse neuroblastoma and myeloma cells, confirming that the IgE-BF produced by this clone shares sequence with expressed IAP elements in different cell types. Thus, information related to the IgE-BF is an integral part of the murine IAP retrotransposon gag gene.

Rodent IgE-binding factor genes are members of an endogenous, retrovirus-like gene family.

Synthesis of IgE by B lymphocytes can be regulated by soluble lymphocyte factors which have affinity for the Fc region of IgE (IgE-binding factors). In previous studies, we identified cDNA clones encoding rodent IgE-binding factors by direct expression in transfected mammalian cells. Here we show that IgE-binding factor cDNA clone 8.3 is a member of the endogenous, retrovirus-like intracisternal A-particle gene family of the mouse. This conclusion is supported by blot hybridization, DNA sequence comparisons, heteroduplex analysis, and immunochemical cross-reactivity of the encoded proteins. The results identify a member of this highly reiterated gene family with a role in regulation of the allergic immune response.

cDNA clones encoding IgE-binding factors from a rat-mouse T-cell hybridoma.

cDNA clones encoding rodent IgE-binding factors (IgE-BF) were isolated from cDNA libraries of a rat-mouse T hybridoma that secretes IgE-suppressive factor (IgE-SF) upon incubation with rat IgE. COS7 cells transfected with two of the cDNAs expressed IgE-BF, which selectively potentiate an in vitro IgE response. IgE-BF expressed in COS7 cells are glycoproteins of approximately equal to 60 and approximately equal to 11 kDa. DNA sequence analysis of an IgE-BF cDNA revealed a 556-amino acid (62 kDa) protein coding region. The results suggest that IgE-potentiating and IgE-suppressive factors share common precursor polypeptides and that the 11-kDa IgE-BF is derived from a 60-kDa precursor.