Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.     

Hybrid immune response to parental liver tissue grafts

Parental-to-F₁-hybrid liver tissue grafts in like-sex donor-recipient combinations survive indefinitely, although several F₁ recipients demonstrate an immunological response to the parental graft. Female F₁ recipients, particularly those carrying theH-2 ^b haplotype, respond vigorously to male parental liver grafts. However F₁ female responses to male parental liver tissue grafts differ substantively from the responses of parental females to syngeneic male grafts. C3H male liver grafts are rejected vigorously by F₁ females as long as the F₁ carries theH-2 ^b haplotype. These findings support previous reports of strong immunological responses to C3H H-Y antigen in female F₁ and C3H.SW animals, a response which is absent in C3H females. Female F₁ hybrids carrying theH-2 ^b haplotype do not reject grafts of B10 or B6 male liver as rapidly as do B10 or B6 parental females. This reduced F₁ response may be related to the formation of hybrid antigens and consequent alteration of the anti-H-Y response. Alternatively, cells that specifically suppress the anti-H-Y response may be present in F₁ hybrids. Factors responsible for suppression appear to be controlled by non-MHC antigens, at least in (OH x B6 or B10) F₁ hybrids.     

Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells

We have produced a series of hybrid IgG₁-IgG_2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differnces between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse γ¹ and γ^2a gene segments. Crossovers occured through out these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H₂L₂ tetramers and properly glycosylated. In addition, all 11 immunoglbulins have identical antigen combining sites specific for the fluorescent hapten ε-dansyll-L-lysine. Protein A binding was used as probe of the structural integrity of the Fc portion of the variant antibodies. Differeneces in protein A binding between IgG₁ and IgG_2a appear to be due to amino acid differances at postions 252 (Thr→Met) and 254 (Thr→Ser) of the heavy chain (EU numbering).     

Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins

The interaction between P-selectin glycoprotein ligand-1/mouse IgG_2b (PSGL-1/mIgG_2b) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Siaα2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG_2b fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 β1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG_2b is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG_2b carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG_2b mucin-type fusion protein carrying Siaα2-3Gal as a multivalent inhibitor of influenza virus is discussed.     

Expression of the neuron-specific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus-mediated cell fusion. The expression of 14-3-2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron-specific 14-3-2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno-types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial-specific protein S-100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.     

Expression and properties of acetylcholine receptors in several clones of mouse neuroblastoma X L cell somatic hybrids

Three clones of somatic cell hybrids between neuroblastoma and L cells, NL-1F, NL-308 and NL-309 (3), have been studied for their electrical excitability and chemosensitivity to acetylcholine (Ach) applied by iontophoresis. Parental and hybrid lines were all treated and tested in media containing mM db-cAMP. The percentage of excitable N X L hybrid cells was as high or higher than that of their neuroblastoma parents. The percentage of cells sensitive to Ach was several-fold higher for the three N X L clones than for the neuroblastoma or L cell parents. While the neuroblastoma parents gave only depolarizing cholinergic responses, the N X L hybrid cells displayed slow hyperpolarizing (H) responses which resembled the H-cholinergic response obtained from L cells. The H-response of the N X L hybrids has properties which indicate the involvement of a muscarinic receptor. A correlation between expression of muscarinic receptors and excitability to electrical current (i.e., action potential ionophores), not found in the neuroblastoma parents, was present in the hybrids. However, a few N X L hybrid cells expressed muscarinic receptors independently from electrical excitability, as is the case for the L cell parent. The three N X L clones are discussed as potentially useful models to study interaction of Ach with muscarinic receptors.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

Specific Features of the Nuclear Genome Recombination in Backcross Progenies of Barley–Wheat Hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42)

The backcross progenies of the barley–wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n= 42) and two alloplasmic lines derived from them were studied using microsatellite markers of barley and wheat. The F₁ hybrids and first backcross plants BC₁ contained the genetic material of both cultivated barley and the cultivars of common wheat involved in developing of these hybrid genotypes. The genomes of BC₃, BC₄, and alloplasmic lines contained no microsatellite markers of the cultivated barley, whereas chromosomes of each homeologous group of common wheat were identified. In chromosomes of backcross progenies BC₃, BC₄, and alloplasmic lines yielded by backcrosses of hybrids and various common wheat cultivars, microsatellite markers of the parental wheat cultivars were shown to undergo recombination.     

Role and strain distribution of genes controlling light chains needed for the expression of an intrastrain cross-reactive idiotype

Several strains of mice were tested for their capacity to provide immunoglobulin L chains required for the expression of the major cross-reactive idiotype (CRI_A) associated with p-azophenylarsonate-specific antibodies of strain A mice. To facilitate testing, mice were bred that were homozygous for Igh-C ^e and Lyt-2 ^a , 3 ^a , i. e., they possessed genes controlling H chains but not L chains required for expression of the CRI_A. Such male mice were mated to females of various strains and their offspring were tested; expression of CRI_A indicated the presence in the female parent of genes controlling the appropriate L chains. All females bearing the Lyt-2 ^a , 3 ^b or Lyt-2 ^b , 3 ^b genotype yielded offspring, most of which were CRI _A ⁺ , whereas all the offspring of females that were Lyt-2 ^a , 3 ^a were CRI _A ^– . The female parents included mice of several strains that are congenic for Lyt-2 ^a , 3 ^b , Lyt-2 ^b , 3 ^b or Lyt-2 ^a , 3 ^b , thus demonstrating very close linkage between the Lyt loci and the expression of CRI_A. In addition, doubly congenic strains of mice with the heavy chain allotype of the CRI _A ⁺ AL/N strain and the Lyt-2 ^a , 3 ^a genotype on a BALB/c background failed to express CRI_A. The data provide further evidence for the similarity of repertoires of L chains in Lyt-3 ^b mice of various strains. When genes were present controlling A/J H chains and L chains of C57BL/6 or BALB/c origin, the quantitative expression of CRI_A was only slightly lower than that observed in A/J mice. Mice possessing genes controlling the H or L chains required for CRI_A expression, but not both, did not express CRI_A but synthesized Ar-specific antibodies which contained low but significant concentrations of the idiotype-associated chain.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Histone gene expression and chromatin structure in mammalian cell hybrids

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

Multigenerational outbreeding effects in Chinook salmon (Oncorhynchus tshawytscha)

Outbreeding, mating between genetically divergent individuals, may result in negative fitness consequences for offspring via outbreeding depression. Outbreeding effects are of notable concern in salmonid research as outbreeding can have major implications for salmon aquaculture and conservation management. We therefore quantified outbreeding effects in two generations (F₁ hybrids and F₂ backcrossed hybrids) of Chinook salmon (Oncorhynchus tshawytscha) derived from captively-reared purebred lines that had been selectively bred for differential performance based on disease resistance and growth rate. Parental lines were crossed in 2009 to create purebred and reciprocal hybrid crosses (n = 53 families), and in 2010 parental and hybrid crosses were crossed to create purebred and backcrossed hybrid crosses (n = 66 families). Although we found significant genetic divergence between the parental lines (F_ST = 0.130), reciprocal F₁ hybrids showed no evidence of outbreeding depression (hybrid breakdown) or favorable heterosis for weight, length, condition or survival. The F₂ backcrossed hybrids showed no outbreeding depression for a suite of fitness related traits measured from egg to sexually mature adult life stages. Our study contributes to the current knowledge of outbreeding effects in salmonids and supports the need for more research to better comprehend the mechanisms driving outbreeding depression.     

Expression of nervous system antigen-3 by lines of mouse fibroblasts and kidney cells and continued expression in hybrid cells

In a survey of the expression on cultured mouse cells of the cell surface antigen known as nervous system antigen-3 (NS-3), it was found that RAG, a renal adenocarcinoma line, expressed that antigen. It was also observed that 3T3, a fibroblast line of unknown tissue origin, expressed NS-3. Cells of these two lines were hybridized with cells of two mouse L cell lines that did not express NS-3. Four hybrid clones were tested for both the 3T3 × L cell cross and the RAG × L cell cross, and all the hybrids were found to be NS-3 positive. All the hybrids had at least 40% as much activity as the NS-3 positive parent. Of the four parental mouse cell lines used, only 3T3 expressed Thy-1.2 antigen on the cell surface. In contrast to the continued expression of NS-3 on hybrid cells, Thy-1.2 antigen was not detectable on two clones of 3T3 × L cell hybrids that were tested.     

Delineation of interspecific epistasis on fiber quality traits in Gossypium hirsutum by ADAA analysis of intermated G. barbadense chromosome substitution lines

Genetic diversity is the foundation of any crop improvement program, but the most cultivated Upland cotton [Gossypium hirsutum L., 2n?=?52, genomic formula?2(AD)₁] has a very narrow gene pool resulting from its evolutionary origin and domestication history. Cultivars of this cotton species (G. hirsutum L.) are prized for their combination of exceptional yield, other agronomic traits, and good fiber properties, whereas the other cultivated 52-chromosome species, G. barbadense L. [2n?=?52, genomic formula?2(AD)₂], is widely regarded as having the opposite attributes. It has exceptionally good fiber qualities, but generally lower yield and less desirable agronomic traits. Breeders have long aspired to combine the best attributes of G. hirsutum and G. barbadense, but have had limited success. F₁ hybrids are readily created and largely fertile, so the limited success may be due to cryptic biological and technical challenges associated with the conventional methods of interspecific introgression. We have developed a complementary alternative approach for introgression based on chromosome substitution line, followed by increasingly sophisticated genetic analyses of chromosome-derived families to describe the inheritance and breeding values of the chromosome substitution lines. Here, we analyze fiber quality traits of progeny families from a partial diallel crossing scheme among selected chromosome substitution lines (CS-B lines). The results provide a more detailed and precise QTL dissection of fiber traits, and an opportunity to examine allelic interaction effects between two substituted chromosomes versus one substituted chromosome. This approach creates new germplasm based on pair wise combinations of quasi-isogenic chromosome substitutions. The relative genetic simplicity of two-chromosome interactions departs significantly from complex or RIL-based populations, in which huge numbers of loci are segregating in all 26 chromosome pairs. Data were analyzed according to the ADAA genetic model, which revealed significant additive, dominance, and additive-by-additive epistasis effects on all of the fiber quality traits associated with the substituted chromosome or chromosome arm of CS-B lines. Fiber of line 3-79, the donor parent for the substituted chromosomes, had the highest Upper Half Mean length (UHM), uniformity ratio, strength, elongation, and lowest micronaire among all parents and hybrids. CS-B16 and CS-B25 had significant additive effects for all fiber traits. Assuming a uniform genetic background of the CS-B lines, the comparative analysis of the double-heterozygous hybrid combinations (CS-B?×?CS-B) versus their respective single heterozygous combinations (CS-B?×?TM-1) demonstrated that interspecific epistatic effects between the genes in the chromosomes played a major role in most of the fiber quality traits. Results showed that fiber of several hybrids including CS-B16?×?CS-B22Lo, CS-B16?×?CS-B25 and CS-B16?×?TM-1 had significantly greater dominance effects for elongation and hybrid CS-B16?×?CS-B17 had higher fiber strength than their parental lines. Multiple antagonistic genetic effects were also present for fiber quality traits associated with most of the substituted chromosomes and chromosome arms. Results from this study highlight the vital importance of epistasis in fiber quality traits and detected novel effects of some cryptic beneficial alleles affecting fiber quality on the 3-79 chromosomes, whose effects were not detected in the 3-79 parental lines.     

Homopoietic histocompatibility (Hh-1) phenotype and the regulation of its expression

Hybrid resistance (HR) is primarily controlled by the genes of the Hemopoietic histocompatibility-1 (Hh-1) locus within the H-2 complex. HR is a consequence of the Hh-1-controlled target determinants in homozygous parental strain mice and their absence in heterozygous F₁ hybrid mice. To examine the mechanism that controls the Hh-1 phenotype, three independent clones of somatic cell hybrids between parental lines EL-4 (C57BL/6 origin, H-2 ^b ) and R1 (C58 origin, H-2 ^k ) were studied. The line EL-4 is Hh-1^b-positive and is subject to HR by H-2 ^b heterozygous F₁ mice, but R1 lacks the Hh-1 ^b allele and is not susceptible to HR. Of the three hybrid clones, F263.2 is Hh-1^b-positive, whereas the other two, F262.2 and F264.2, are Hh-1-negative, as judged by these cells' capacity to compete in vivo with the grafted parental C57BL/6 bone marrow cells in the resistant (C57BL/6 × C3H)F₁ mice. All three clones express the H-2^b and H-2^k class I antigens equally well, are susceptible to activated NK cells to the same extent, and all carry four copies of chromosome 17. However, Southern analysis reveals that clone F263.2 contains three copies of H-2 ^b chromosome and one H-2 ^k , whereas the other two clones carry two copies each of the parental chromosome 17. The results suggest that the relative copy number of specific alleles is the crucial determinanr of the Hh-1 phenotype, and render unlikely both the gene dosage hypothesis and the trans-acting dominant suppression hypothesis to account for the noncodominant expression of the Hh-1 phenotype.     

Phenotypic and molecular expression of albumin in rat hepatoma X L cell hybrids

A rat hepatoma cell line (H₄A_ZC₂) was characterized with respect to seven liver-specific phenotypes. Ten clones from the fusion of H₄A_ZC₂ and mouse L cell were analyzed for the expression of these phenotypes. The only hepatic function retained by the hybrid clones was rat albumin synthesis which continued at reduced levels relative to the hepatoma parent. Rat albumin cDNA analysis of RNA from parental and hybrid cells indicated that the reduction in albumin production observed in the hybrids was reflected in coordinate reduction of cytoplasmic rat albumin mRNA.