HAT Medium Research Articles

A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies

A simple method is described by which hybridomas can be selected and cloned in a single step immediately after fusion. This is done by plating the cells in semi-solid medium containing methylcellulose and the components of the HAT selection system. A number of variables have been examined in order to optimise the technique. The system is particularly suitable for isolating large numbers of hybridomas secreting different monoclonal antibodies. Evidence is presented to show that the colonies which grow in the system are in all probability clones. Thus, the need for routine recloning of the hybridomas is eliminated. In this way, the technique cuts down on the amount of tissue culture work associated with the production of monoclonal antibodies. Using this technique, it is easier to plate out large numbers of cells and to recover many independent hybridoma clones, than is the case when using cloning by limiting dilution.

Appearance of double minute chromosomes in somatic cell hybridisation experiments involving the hat selection system

In somatic cell hybridisation experiments using the HAT selection system, parental HPRT − cells have been shown to escape selection. These cells contain varying numbers of double minute chromosomes which are probably cytological consequences of amplification of the dihydrofolate reductase structural genes. When the double minute containing cells were challenged with varying concentrations of antifolate drug the number of double minutes per cell correlated well with the aminopterin concentration in the culture medium. The implications of these findings for cell fusion and gene transfer experiments is discussed.

Coordinate expression of parietal endodermal functions in hybrids of embryonal carcinoma and endodermal cells.

A derivative, FOT5, of the F9 murine embryonal carcinoma cell line which is resistant to ouabain and thioguanine was fused with a near diploid parietal endodermal cell line, PFHR9, Hybrid clones (ENEC1 to ENEC5) were isolated in HAT Medium containing ouabain at a frequency of approximately 2 x 10(-4). The DNA contents and chromosome number of the ENEC hybrids were approximately the sum of those of the parents. Five hybrid cell lines examined in detail expressed the following parietal endodermal functions: plasminogen activator activity, basement membrane proteins, and endodermal cytoskeletal proteins. Embryonal carcinoma characteristic functions (tumorigenicity, a stage specific embryonic antigen, and high alkaline phosphatase activity) were extinguished in the hybrids. No hybrid clones with embryonal carcinoma morphology were observed among 1,358 hybrid clones examined. Hybrids, propagated for over 100 generations, continued to express endodermal functions and not embryonal carcinoma functions. The coordinate expression of endodermal functions and the extinction of embryonal carcinoma functions in the ENEC hybrids suggest that the parietal endodermal cells contain diffusible activities which extinguish embryonal carcinoma functions and possibly cause the embryonal carcinoma genome to express parietal endodermal characteristics.

Coordinate expression of parietal endodermal functions in hybrids of embryonal carcinoma and endodermal cells.

A derivative, FOT5, of the F9 murine embryonal carcinoma cell line which is resistant to ouabain and thioguanine was fused with a near diploid parietal endodermal cell line, PFHR9, Hybrid clones (ENEC1 to ENEC5) were isolated in HAT Medium containing ouabain at a frequency of approximately 2 x 10(-4). The DNA contents and chromosome number of the ENEC hybrids were approximately the sum of those of the parents. Five hybrid cell lines examined in detail expressed the following parietal endodermal functions: plasminogen activator activity, basement membrane proteins, and endodermal cytoskeletal proteins. Embryonal carcinoma characteristic functions (tumorigenicity, a stage specific embryonic antigen, and high alkaline phosphatase activity) were extinguished in the hybrids. No hybrid clones with embryonal carcinoma morphology were observed among 1,358 hybrid clones examined. Hybrids, propagated for over 100 generations, continued to express endodermal functions and not embryonal carcinoma functions. The coordinate expression of endodermal functions and the extinction of embryonal carcinoma functions in the ENEC hybrids suggest that the parietal endodermal cells contain diffusible activities which extinguish embryonal carcinoma functions and possibly cause the embryonal carcinoma genome to express parietal endodermal characteristics.

Proteins containing only half of the coding information of early region 1b of adenovirus are functional in human cells transformed with the herpes simplex virus type 1 thymidine kinase gene and adenovirus type 2 DNA.

We introduced into tk- human 143 cells adenovirus type 2 (Ad2) genes by transformation with a plasmid (p711) containing both Ad2 sequences and the herpes simplex virus type 1 (HSV-1) tk gene. p711 contained approximately the left 8% of the Ad2 genome inserted in the HindIII site of pBR322, whereas the fragment of HSV-1 containing the tk gene was inserted in the BamHI site. Three tk+ cell lines were isolated after selection in HAT medium. The arrangement of viral sequences in the three transformants was analyzed by restriction endonuclease digestion and filter hybridization. All three lines contained a single insertion of Ad2 DNA which was present at approximately one copy per cell. The arrangement of Ad2 sequences in these lines was identical to that found in the linear p711 DNA used in the transformation. S1 analysis of the Ad2-specified RNA from two of these lines indicated that the early region 1a mRNA's were synthesized, though in lower amounts than found in lytic infections. These cell lines contained only the left half of early region 1b (4.6 to 11.2), which encoded the 5' portion of the 1b mRNA's. A complex pattern of 1b RNAs was made in these cell lines. Transcription of most of these RNAs began at or near the 1b promoter and proceeded through the 1b sequences into the flanking pBR322, HSV-1, or host sequences. Since many of the RNAs were terminated or spliced in the HSV-1 (anti-sense strand) or pBR322 sequences, new RNA processing sites must be used in the formation of these mRNA's. All three lines fully complemented the 1a deletion mutant Ad5 dl312. Surprisingly, these lines also permitted the growth of 1b deletion mutants (Ad5 dl313 and Ad5 dl434), although the complementation was not always complete. Presumably the new gene product(s) which contained only part of the 1b gene provided most of the essential function(s) required for viral multiplication. Alternatively, the 1b 19-kilodalton protein which was entirely encoded by the adenovirus sequences present in these cell lines was sufficient for viral growth even in the absence of the 1b 55-kilodalton protein.

Effect of temperature on mutation in cultured human skin fibroblasts

Because clothing enhances the gonadal temperature in the human male and because as a consequence of that the spontaneous mutation rate might be increased, a study was undertaken to determine the effect of temperature on mutation towards HPRT deficiency in human diploid skin fibroblasts. Culturing of the cells in HAT medium containing azaserine, to remove pre-existing mutants, was highly mutagenic. Some results suggested that azaserine acts as an indirect mutagen. The mutational process in the presence of azaserine was influenced by temperature, a rise in temperature from 33 to 37°C leading to a more than 10-fold increase in mutation rate per cell generation. This temperature dependence, taken as a starting point for the estimation of the consequences of the rise in gonadal temperature, could be responsible for an increase of 135% in the incidence of a sex-linked lethal (Lesch-Nyhan) in the human population. Epidemiological data on the frequency of Lesch-Nyhan disease and on the ratio of paternal and maternal mutations leading to Lesch-Nyhan disease do not contradict the findings in the cultured cells.

Lymphotoxin and immune (gamma) interferon production by T cell lines and hybrids.

The goal of our work is an in-depth analysis of the thymus derived lymphocyte (T cell) and its products. T cell functions, products and molecular biology can now be analyzed through the use of continual T cell lines and hybrids. Primary T cells can be. immortalized within the milieu of a proliferating AKR Thy 1.1+ HGPRT- lymphoma, BW 5147, through the technique of somatic cell hybridization and selection in HAT medium. Constitutive traits such as isozymes, Thy 1 and H-2 antigens of both parents, and antigen specific activity of the primary parent can be expressed by such hybrids (1, 2, 3), and expression of many such activities is regulated by culture phase (4). We (5) and others (6, 7) have demonstrated constitutive production of antigen specific suppressor factors by such hybrids. However, it is not always possible to obtain a T cell hybrid that maintains expression of all functions of the primary parent, as chromosomes may segregate from T cell hybrids with concomitant loss of traits. This situation also occurs in B cell hybridomas (8) and all other types of somatic cell hybrids (9).

Stimulation of cytolytic T lymphocytes by azaguanine-resistant mouse tumor cells in selective HAT medium

Primed syngeneic or unprimed allogeneic mouse spleen cells were stimulated with azaguanine-resistant P815 tumor cells that were killed by the addition of aminopterin to the stimulation medium. The recovery of lymphocytes and their cytolytic activity and specificity were similar to those obtained after stimulation with irradiated cells. This method conveniently replaces the inactivation of stimulatory cells by irradiation or mitomycin treatment. Moreover, it has the advantage of inactivating not only the stimulatory cells but also the tumor cells that often contaminate the spleens of tumor-bearing animals, provided these animals have been inoculated with azaguanine-resistant tumor cell mutants.

Cell fusion-induced mouse neuroblastomas HPRT revertants with variant enzyme and elevated HPRT protein levels.

Fusion of 6-thioguanine-resistant mouse neuroblastoma to HeLa whole and minicells generated neuroblastoma HPRT revertants in addition to true cell hybrids. All revertants contained HPRT with decreased electrophoretic mobility and heat stability relative to wild-type mouse enzyme. Revertant HPRT expression was dependent on continuous HAT selection. Quantitative immunoadsorption experiments showed that revertants with low HPRT specific activity had wild-type levels of HPRT protein while a revertant with high apparent activity (NBR4) contained elevated levels of variant protein. HPRT extracted from NBR4 had decreased affinity of both hypoxanthine and PRPP relative to wild type. Evidence is presented that HPRT elevation is dependent on the reversion process itself.

Interference in hybrid clone selection caused by Mycoplasma hyorhinis infection

Mycoplasma hyorhinis strains were isolated from Chinese hamster DON cells which lacked the ability to produce hybrid colonies in HAT medium. The mycoplasma isolates were virtually devoid of HGPRT activity in vivo and in vitro in the presence of excess co-enzyme, phosphoribosylpyrophosphate. Deliberate infection of mycoplasma-free cells caused no alterations in the HGPRT − and TK − phenotypes of the cells. Heterokaryon formation with infected cells was normal and the failure to produce hybrid colonies resulted from depletion, by nucleoside phosphorylase activity, of exogenous thymidine required for rescue of hybrid cells in HAT medium. Increasing the thymidine concentration and repeatedly replenishing HAT medium permitted hybrid clone formation.

Linkage and expression of foreign DNA in cultured animal cells.

The ability to introduce defined genes into cultured cells radically alters the prospects for understanding differentiation and growth control in higher organisms. Stable transformation of animal cells may be effected by the delivery of DNA as a calcium phosphate coprecipitate (Graham and van der Eb 1973). Since the uptake and expression of DNA is a relatively rare event, transformants are usually isolated by selection for cells that have acquired a new phenotype. For this purpose the thymidine kinase (tk)1 gene of herpes simplex virus type 1 (HSV-1) is often used as the selectable vector with tk+ recipient cells (Wigler et al. 1977). The tk+ transformants are then selected in HAT medium. Nonselectable genes may be stably introduced into cells either by direct ligation to the selectable vector (Mantei et al. 1979) or by unlinked cotransformation (Wigler et al. 1979b).

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate antigenic changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion, myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitatively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.

Culture of sheep X mouse hybridoma cells in vitro.

Hybridomas were made between NS1/1-Ag4-1 mouse myeloma cells and spleen and lymph node cells from a sheep immunized with sheep red cells (RBC). The hybrid colonies grew well in culture but there was a substantial loss of sheep chromosomes. No hemolytic or agglutinating antibodies were detected in the culture supernatants after the 17th day following fusion, but immunofluorescence tests indicated that a few of the cells may have been expressing sheep IgG. Cytogenetic comparison of cells grown with and without HAT medium provided evidence that the enzyme HGPRT is located on the X chromosome of sheep as it is in man and mouse. Hybridoma isozyme patterns of esterase, G6PD, 6PGD, NP, LDH and SOD tested between the 63rd and 71st day of culture were like those of NS1; NP and LDH also showed zones that probably came from the sheep component.

Monoclonal antibodies to guinea pig Ia antigens. I. Production, serologic, and immunochemical characterization.

Xenogeneic antibodies to guinea pig Ia antigens were produced by application of the hybridoma technique. BALB/c mice were immunized with the Ia-positive B cell leukemia line EN-L2C of strain 2 guinea pigs, and the spleen cells were fused to the mouse myeloma line NS-1. Culture supernatants of hybrids growing in the selective HAT medium were screened in a binding assay, and hybrids selectively reactive with EN-L2C but not with its Ia-negative variant BZ-L2C were cloned. Ascitic fluid was produced and used as a source of antibody. Four independently derived antibodies proved to be specific for Ia antigens. Two of the antibodies (25E3 and 25E11) seemed to recognize alloantigenic determinants on strain 2 Ia antigens in that they bound only to the cells of strain 2 but not to the cells of strain 13 animals. Two other antibodies (22C4 and 27E7) were found to react with both strain 2 and strain 13 cells, and immunoprecipitation experiments revealed that these antibodies were directed to common determinants shared between certain strain 2 and strain 13 Ia antigens. All antibodies were cytotoxic in the presence of complement for EN-L2C (but not for BZ-L2C) leukemia cells and for lymph node, spleen, peritoneal exudate cells, thymocytes, and purified T cells of strain 2 guinea pigs. In addition, 22C4 was cytolytic for cells of both strains. These results indicate that antibodies can be obtained through the hybridoma technique that are able to distinguish different determinants on Ia antigen molecules of te guinea pig. These antibodies should provide a useful tool for further study of the role of Ia antigens in cellular interaction and Ia-Ir gene function.

High efficiency transformation by direct microinjection of DNA into cultured mammalian cells

Direct microinjection of DNA by glass micropipettes was used to introduced the Herpes simplex virus thymidine kinase gene into cultured mammalian cells. When DNA was delivered directly into the nuclei of LMTK −, a mouse cell line deficient in thymidine kinase activity, 50–100% of the cells expressed TK enzymatic activity. In contrast, no TK activity could be detected when the DNA was injected into the cytoplasm. The number of injected LMTK − cells capable of indefinite growth in a TK + selective medium (that is, transformants) depended on the nature of the plasmid DNA into which the HSV-TK gene was inserted. One cell in 500–1000 cells which received nuclear injections with pBR322/TK DNA gave rise to a viable colony when grown in HAT medium (that is, a TK + selective medium). The transformation frequency increased to one in five injected cells when specific SV40 DNA sequences were also introduced into the HSV-TK plasmid. With the microinjection procedure transformation frequency was relatively insensitive to DNA concentration and did not depend on co-injecting with a carrier DNA. Most of the transformants were stable in nonselective medium as soon as they could be tested.

Association of the herpes simplex-1 viral gene for thymidine kinase with the human gene for adenylate kinase-1 in biochemically transformed cells.

The herpes simplex type 1 biochemically transformed human cell line, HB-1, was fused with thymidine kinase deficient rodent cells, and 18 hybrids were isolated using the HAT-ouabain selection system. The selected enzyme, viral thymidine kinase, was present in all 18 hybrids. In 16 of 18 hybrids the viral gene for thymidine kinase cosegregated with the human gene for adenylate kinase-1 (AK-1). Thirty-six bromodeoxyuridine (BrdUrd) resistant sublines were isolated from the 16 human AK-1 positive hybrids. Each BrdUrd-resistant subline was examined for the presence of the viral TK gene by back-selection in HAT medium, and for human AK-1. In all 36 BrdUrd-resistant sublines the viral TK gene cosegregated with the human AK-1 gene. These results indicate that the transforming viral DNA fragment was associated with a specific human chromosomal region in HB-1 cells.

Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA

We have introduced adenovirus 2 genes into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene. Tk+ cells were isolated after selection and maintenance in HAT medium. Several co-transformed lines are able to complement the growth of Ad5 dl312 (delta 1.2--3.7) and Ad5 dl434 (delta 2.6--8.7), deletion mutants that lack sequences from the left end of the viral genome. The amount and arrangement of viral sequences in the co-transformed cell lines have been analyzed by restriction endonuclease digestion and filter hybridization. Most of the cell lines contain a single insertion of the HSV-1 tk fragment and a single insert of adenoviral DNA. However, one line (B1) contains at least four different insertions, two of which are present in multiple copies. The adenoviral DNA in all cell lines is composed of sequences from the left end of the genome and extends for varying lengths in different lines. Two cell lines that complement deletion mutants efficiently synthesize both early region 1a and 1b mRNAs. The B1 line synthesizes low levels of 1a mRNA, higher levels of 1b mRNA and a unique mRNA that maps to the right of the 1b gene family. When grown continuously in HAT medium, some cell lines are quite stable while others are fairly unstable. Some tk+ subclones support the growth of viral mutants as well as the parental line while others give reduced levels of complementation. For all tk+ subclones examined, the alteration or reduction in viral gene expression is independent of changes in the pattern of integration of viral DNA.

Assignment of a human genetic locus to chromosome 5 which corrects the heat sensitive lesion associated with reduced leucyl-tRNA synthetase activity in ts025Cl Chinese hamster cells.

A heat-sensitive (hs) leucyl-tRNA synthetase (leuRS) deficient CHO mutant, ts025Cl, was fused with human leukocytes and hybrids isolated in HAT medium at the nonpermissive temperature. Nineteen heat-resistant (hr) and 14 hs subclones were isolated from four independent primary hybrids and tested for the expression of 24 human isozymes which have been assigned to 17 human chromosomes. Four hr independent subclones and three hs independent subclones were analyzed for the presence of human chromosomes. A pattern of concordant segregation was noted for the hr phenotype, human hexosaminidase B (EC 3.2.1.30) and human chromosome 5. Based on these results, we have defined the human genetic locus which corrects the heat-sensitive lesion in ts025Cl as hr025Cl and have assigned this locus to human chromosome 5. Two hr hybrids exhibited leuRS activity 2.5 and 4 times the leuRS activity of ts025Cl but a wild-type level of activity was not restored. One hs hybrid had only 73% of the leuRS activity exhibited by ts025Cl while another hs hybrid had 1.8 times the leuRS activity of ts025Cl.

Differentiation of thymidine kinase deficient mouse myoblasts in the presence of 5′-bromodeoxyuridine

The hypothesis that 5′-bromodeoxyuridine (BUdR) inhibits differentiation via incorporation into DNA was examined with a strain of thymidine kinase-deficient (TK −) mouse myoblasts. TK − cells were derived from a permanent clonal line of mouse myoblasts which were mutagenized with ethylmethanesulfonate and then carried through 3 BUdR/visible light selection cycles. Cells were characterized as TK − by their inability to grow in HAT medium, their minimal ability to incorporate [ 3H]thymidine into DNA, and the absence of all but trace amounts of thymidine kinase in cell extracts. TK − isolates were initially unstable with respect to myogenic properties, but a stable diploid myogenic TK − strain was obtained after several serial subclonal cultivations. To monitor effects of BUdR on muscle differentiation, colonies of proliferating wild-type and TK − myoblasts were incubated with or without BUdR for 24 h. Differentiation was then induced by incubating the cultures in mitogen-depleted medium for 36 h. Exposure of wild-type myoblasts to 5 or 15 μM BUdR caused 84 and 98% inhibition of myotube formation, whereas exposure of TK − myoblasts to these BUdR levels caused only 15 and 25% inhibition. Similar results were obtained when differentiation was assessed by measuring creatine phosphokinase activity. These results indicate that BUdR must be metabolized to at least the mono-phosphate form to inhibit muscle differentiation, and they are consistent with the hypothesis that BUdR acts via incorporation into DNA.

Branched-chain aminotransferase deficiency in Chinese hamster cells complemented by two independent genes on human chromosomes 12 and 19

Branched-chain aminotransferase (BCT) catalyzes the reversible transamination of the branched-chain alpha-keto acids to the branched-chain L-amino acids. Since branched-chain L-amino acids (L-isoleucine, L-leucine, and L-valine) are essential for cell growth, cells which lack BCT were unable to proliferate in media containing alpha-keto acids in place of the corresponding L-amino acids. CHW-1102, a Chinese hamster cell line, lacks BCT and does not grow in alpha-keto acid media. Somatic cell hybrids were made by the fusion of CHW-1102 (HPRT-) with several human cell lines and isolated on HAT medium. Growth assays of hybrid clones on alpha-keto acid selection media independent of the HAT selection medium indicated two cell hybrid phenotypes: either (1) the hybrid clone, like the parental CHW-1102, could not utilize alpha-keto acid media, or (2) the hybrid could proliferate on all three alpha-keto acid media. The ability of hybrid cells to proliferate on alpha-keto acid media correlated with the presence of either of two human genes which independently complemented the Chinese hamster deficiency. Two human genes. BCT1 assigned to chromosome 12 and BCT2 assigned to chromosome 19, were demonstrated to code for the expression of two molecular forms of BCT.