Immunoglobulin production by a human-mouse somatic cell hybrid

The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobulin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.     

Sequence of centromere separation: Separation in a quasi-stable mouse-human somatic cell hybrid

A quasi-stable mouse-human hybrid cell line, HR61, containing between one and ten human chromosomes was analyzed for the sequence of centromere separation. The purpose was to determine which genome of the two initiates centromere separation first. The data clearly indicate that the separation of centromeres of the human genome is not only initiated but is completed before any centromeres from the mouse chromosomes start splitting into daughter units. The information on whether uniparental chromosome loss results from a lack of deposition of kinetochore proteins was equivocal. The human genome also completes its DNA replication before the mouse genome does. Our studies, therefore, show that the timing of centromere separation is tightly linked to the completion of replication of DNA. At least in this cell line the segregant genome is not the one which exhibits delayed DNA replication.     

Role of permanent dicentric systems in carrot somatic embryogenesis

Summary A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.     

Regulation of species-specific ribosomal RNA in a somatic hybrid cell.

When 13B hamster-mouse hybrid cells are harvested either right after 4 h of incubation with [me-3H]methionine or following 26 h of "chase" with excess non-radioactive methionine, in both cases about half of the labeled cytoplasmic rRNA is of hamster type. It had been previously shown in this laboratory (Eliceiri, G.L. (1973) Biochim. Biophys. Acta 312, 737-741) that when [3H]uridine was the radioactive precursor about 80% of the labeled cytoplasmic rRNA was of hamster type after a short incubation, and about half after a long incubation. It is postulated that a temporary difference in the specific acitivity of [3H]UTP in possibly segregated mouse and hamster types of nucleoli might account for these results. The master/mouse ratio of cytoplasmic rRNA in hybrid 13B is similar in free and in membrane-bound ribosomes, and in ribosomes of sparse (rapidly growing) cell populations and of confluent (slowly growing) cells.     

Characterization of a zebrafish/mouse somatic cell hybrid panel

We have characterized a collection of zebrafish/mouse somatic cell hybrids with 211 genes and markers chosen from the 25 zebrafish linkage groups. Most of the zebrafish genome is represented in this collection with 88% of genes/markers present in at least one hybrid cell line. Although most hybrids contain chromosomal fragments, there are a few instances where a complete or nearly complete zebrafish chromosome has been maintained in a mouse background, based on multiple markers covering the entire chromosome. In addition to their use in mapping studies, this collection of somatic cell hybrids should constitute an important tool as a source of specific chromosome fragments and for assessing the function of genome regions.     

Genetic engineering of a mouse: Dr. Frank Ruddle and somatic cell genetics

Genetic engineering is the process of modifying an organism's genetic composition by adding foreign genes to produce desired traits or evaluate function. Dr. Jon W. Gordon and Sterling Professor Emeritus at Yale Dr. Frank H. Ruddle were pioneers in mammalian gene transfer research. Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring. Transgenic mice have revolutionized biology, medicine, and biotechnology in the 21st century. In brief, this review revisits their creation of transgenic mice and discusses a few evolving applications of their transgenic technology used in biomedical research.     

Characterization of a depolarizing dopamine response in a vertebrate neuronal somatic cell hybrid

The physiology and pharmacology of a depolarizing dopamine response was studied in the vertebrate neuronal somatic cell hybrid TCX11. The average resting membrane potential was ?50 mV (S.D. = ±7) with a membrane resistance of 40.5 mOhms (S.D. = ±8) as determined from intracellular recordings. Depolarizing current pulses did not elicit an action potential. Cells displayed a linear current-voltage relationship when artificially depolarized up to +30 mV. Iontophoretically applied dopamine elicited a depolarizing response with a conductance increase and a reversal potential of ?15 mV (S.D. = ±4.7). Experiments altering medium ion concentrations demonstrated the conductance increase was to sodium and most likely potassium. The dopamine agonist ET495 (Piribedil) and the analogue epinine mimicked dopamine, while closely related biogenic amines, with the exception of noradrenaline, elicited no response. Apomorphine also elicited a depolarizing response but was much less efficacious than Piribedil. Noradrenaline was less potent than dopamine and appeared to act at the dopamine receptor. Methylation (3-methoxytyramine) or absence of the 3-hydroxy group (tyramine) of dopamine resulted in total loss of activity. The dopamine antagonists chlorpromazine, trifluoperazine, promazine, and bulbocapnine reversibly blocked the response to dopamine at medium concentrations less than 5 μM. The adrenergic antagonist phentolamine blocked the response while phenoxybenzamine only reduced the response at higher concentrations. The acetylcholine antagonists α-bungarotoxin, hexamethonium, and scopolamine did not block the dopamine response. Both d-tubocurarine and atropine acted as antagonists. Collectively, these results demonstrate the presence of a receptor on a cultured cell line that is specific for dopamine, mediates a depolarizing and conductance increase response to dopamine, and displays the pharmacology most closely associated with dopamine receptors.     

Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.     

Establishment of a human somatic hybrid cell line for recombinant protein production

Cell fusion techniques were used to derive mammalian host cell lines suitable for large-scale production of therapeutic proteins. Although the 293S cell line, of human embryonic kidney origin, is an excellent host cell for mammalian gene expression, these cells have a tendency to form large and tight aggregates in suspension cultures and bioreactors. To solve the problem of aggregation, 293S cells were fused to a human suspension cell line, 2B8 (a Burkitt's lymphoma derivative), using polyethylene glycol (PEG). The PEG-treated 293S and 2B8 cells were selected in a medium supplemented with hypoxanthine-aminopterin-thymidine and G418 (1 mg/ml) to eliminate nonfused cells. These hybrid clones, designated as HKB (hybrid of kidney and B cells), are negative for endogenous immunoglobulin expression. Most clones are readily adaptable to serum-free suspension culture under shaking conditions without forming large and tight aggregates. One clone, HKB11, was shown to support high-level expression of cytokines [interleukin (IL)-2 and IL-4], ICAM-1 and rFVIII in a side-by-side comparison with 293 and Chinese hamster ovary cells. The above-described characteristics of HKB cells indicate that HKB11 is a favorable cell host for the production of human therapeutic proteins.     

Human lymphocyte-mouse myeloma somatic cell hybrids: selective hybrid formation.

Fusion of unfractionated human lymphocytes with mouse myeloma cells resulted in proliferating hybrid colonies, almost all producting human Ig. We examined whether this high frequency of Ig production was the result of selective formation of human B lymphocyte-mouse myeloma hybrids, rather than induction of Ig genes in T lymphocytes. Unfractionated peripheral lymphocytes and B lymphocytes from patients with the common variable form of agammaglobulinemia formed proliferating somatic cell hybrid colonies. In contrast, peripheral lymphocytes from a patient with agammaglobulinema who lacked B lymphocytes, as well as albumin gradient fractions of peripheral blood which do not contain B lymphocytes, failed to produce somatic cell hybrids with three different myeloma parent cell lines. B, T, and precursor lymphocytes all had Sendai virus receptors, as witnessed by viral agglutination. We conclude that fusion of human lymphocytes with mouse myeloma cells results in selective hybrid formation, rather than activation of Ig genes in disparate cell types. Only B lymphocyte-mouse myeloma heterokaryons form hybrid cells.