Rapid cloning of mammalian cells with honeycomb cloning plates and nonlethal vital stains

A rapid and technically simple method for cloning both adhesive and nonadhesive mammalian cells is described. The procedure employs (a) honeycomb cloning plates and (b) nonlethal vital stains. Instead of placing cloning rings around colonies, cells are initially seeded at clonal density directly into a plate containing an array of cloning rings (the honeycomb plate). Hence, the time involved in placing cloning rings around colonies is eliminated. Second, clone-containing wells of the honeycomb plate are easily identified by staining plates with the nonlethal vital stains, MTT or INT tetrazolium. Vital staining eliminates the time involved in searching for clones. Last, clones are transferred with a cotton-tipped swab thereby eliminating the time involved in trypsinization of cells. In this fashion, one can pick and transfer clones of substrate adherent mammalian cells at a rate of one clone/10 to 15 s. Thus, mammalian cells can be cloned as rapidly as cloning can be carried out in microbial systems.     

Frontiers in mammalian cells culture

Summary For the past 60 years, fundamental discoveries in eukaryotic biology using mammalian cell cultures have been significant but modest relative to the enormous potential. Combined with advances in technologies of cell and molecular biology, mammalian cell culture technology is becoming a major, if not essential tool, for fundamental discovery in eukaryotic biology. Reconstruction of the milieu for cells has progressed from simple salt solutions supporting brief survival of tissues outside the body to synthesis of the complete set of structurally defined nutrients, hormones and elements of the extracellular matrix needed to reconstruct complex tissues from cells. The isolation of specific cell types in completely defined environments reveals the true complexity of the mammalian cell and its environment as a dynamic interactive physiological unit. Cell cultures provide the tool for detection and dissection of the mechanism of action of cellular regulators and the genes that determine individual aspects of cell behavior. The technology underpins advances in virology, somatic cell genetics, endocrinology, carcinogenesis, toxicology, pharmacology, hematopoiesis and immunology, and is becoming a major tool in develomental biology, complex tissue physiology and production of unique mammalian cell-derived biologicals in industry. This article is the first of a series of invited reviews aimed at identifying fundamental contributions and current challenges associated with research activities in subdiscriplines of cell and developmental biology in vitro. This treatise is dedicated to Dr. Brian Kimes, Program Director at the National Cancer Institute, whose vision, encouragement and support have contributed significantly to modern developments in mammalian cell culture.     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

Isolation of glucagon-secreting cell lines by cloning insulinoma cells

Summary Six glucagon-secreting cell lines designated as In-R1G1,-G3,-G7,-G9,-G10, and-G11 were isolated from insulinoma cells (In-111-R1) by single cell cloning. A small amount of insulin was also detectable in the incubation medium when hormone secretion was stimulated by the addition of arginine or theophylline. These cell lines grew as monolayers and the population doubling times varied from 16.8 to 28.8 h. Karyologically these clones were aneuploid and the modes of chromosome numbers were 61 to 70. Electron microscopic examination of one of these clones showed that these cells contained moderately developed Golgi apparatus and a few secretory granules, which more or less resembled α-cell granules. By gell filtration study of the incubation medium, glucagon and glucagonlike material were eluted. The molecular weight of the latter was approximately 9000, which suggested the concomitant secretion of rpoglucagon into the medium. The levels of secreted glucagon in basal state were 0.3 to 3.0 ng/10⁶ cells/2 h. Glucagon secretion was markedly enhanced in the presence of amino acids. Glucagon secretion increased slightly in the presence of high concentration of glucose in Hanks' balanced salt solution; however it was not affected by the varying concentrations of glucose when the cells were incubated in complete media with amino acids. Glucagon secretion was also stimulated by the addition of theophylline. These clonal cell lines seem to provide a useful tool for investigating the mechanism of glucagon secretion.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning, which is based on human somatic cell nuclear transfer, is one of our major research objectives. Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos, the effects of type, passage, and preparation method of donor cells on embryo development remain unclear. In our experiment, cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell, skin fibroblast, and cumulus cells. The cumulus cell embryos showed significantly higher development rates than the other two (P < 0.05). The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference. Also, fluorescence in situ hybridization (FISH) was conducted to detect nuclear derivation of the embryos. The result showed that the nuclei of the inter-species cloned embryo cells came from human. We conclude that (1) cloned embryos can be constructed through human-rabbit interspecies nuclear transfer; (2) different kinds of somatic cells result in different efficiency of nuclear transfer, while in vitro passage of the donor does not influence embryo development; (3) refrigeration is a convenient and efficient donor cell preparation method. Finally, it is feasible to detect DNA genotype through FISH. Translated from Zoological Research, 2005, 26(4): 416–421 [译自: 动物学研究]     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

Mutations and epimutations in mammalian cells

Early studies on heritable variation in cultured mammalian cells suggested that both mutation and epigenetic events might be involved. The importance of mutations has subsequently been fully documented, but only recently has an alternative form of inheritance been uncovered. This is based on the post-synthetic methylation of cytosine in regulatory regions of genes. The pattern of methylation is heritable, and in almost all cases studied, methylation of a region is associated with lack of gene expression. Such silent genes can be reactivated by the powerful demethylating agent 5-azacytidine (5-aza-CR). Changes in heritable DNA methylation which alter phenotype are referred to as epimutations. It now seems very likely that the well known ‘functional hemizygosity’ in CHO cells and other near diploid cell lines is due to the existence of one active and one silent gene at many autosomal loci. It is clear that permanent cell lines inactive genes by de novo methylation, whereas normal diploid cells do not have this activity. This has important implications for our understanding of cellular transformation, tumor progression, and the increase in chromosome number frequently associated with these cellular changes. It is likely that both mutations and epimutations are important in the emergence of fully transformed tumorigenic cells. Agents which increase or reduce DNA methylation in cells can be regarded as epimutagens, although in many cases the mechanisms of inducing hypo- or hyper-methylation are not understood. Two exceptions are 5-aza-CR which inhibits the normal DNA maintenance methylase activity, and 5-methyldeoxycytidine triphosphate which is incorporated into cellular DNA following electroporation and has been shown to silence genes.     

Hydroxyurea-resistant mouse L cells with elevated levels of drug-resistant ribonucleotide reductase activity

We describe the isolation and partial characterization of a mouse L-cell line which is resistant to normally highly cytotoxic concentrations of hydroxyurea. A detailed analysis of the target enzyme ribonucleotide reductase in both wild-type and hydroxyurea-resistant enzyme preparations suggests that the drug-resistant cells form a ribonucleotide reductase enzyme which contains a structural alteration, rendering it less sensitive to inhibition by hydroxyurea. K₁ values for hydroxyurea inhibition of ribonucleotide reduction in enzyme preparations from hydroxyurea-resistant cells were significantly higher than corresponding values from preparations from wild-type cells. The K_m for CDP reduction in enzyme preparations of drug-resistant cells was approximately threefold higher than the corresponding parental wild-type value. In addition, in vivo enzyme assays detected a major difference between the temperature profiles of ribonucleotide reduction in nucleotide-permeable drug-resistant and wild-type cells. When levels of ribonucleotide reductase activity were measured in vivo, it was found that the drug-resistant cells contained approximately 3 times the wild-type level of CDP reductase activity and twice wild-type level of GDP reductase activity. This combination of enhanced enzyme levels plus an altered sensitivity to drug inhibition can easily account for the drug-resistance phenotype. The properties of these hydroxyurea-resistant cells indicate that they will be useful for genetic and biochemical studies.This work was supported by the N.S.E.R.C. of Canada and the Muscular Dystrophy Association of Canada through research funds (J. A. W.) and by the N.R.C. of Canada through a graduate scholarship (B. A. K.).     

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes

ABSTRACT

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.     

Presence of an immunologically-defined class of peri-Golgi vesicles in cultured mammalian cells

Immunsera of mice injected with clathrin-depleted coated vesicle membranes, purified from rat liver, revealed a preferential labeling of some perinuclear structures by immunofluorescence microscopy on NRK cells. Subsequent production of 4 monoclonal antibodies was achieved. The antigen was strictly located in the Golgi area of the cell but was not an intrinsic element of the Golgi complex. The restricted location of the structures excluded these were lysosomes which appeared more dispersed in these cells. After nocodazole treatment the material was found dispersed in the cytoplasm. This provided a means of distinguishing the antigen from clathrin-coated structures and Golgi intrinsic elements. Immunolocalization at the electron microscope level confirmed the data obtained at the light level. Some peroxidase reaction product was rarely associated with Golgi elements, but predominantly stained small neighboring Golgi vesicles (50 nm diameter), as well as tubulo-elongated structures and some large (500 nm) irregular-shaped vesicles. A 32 kDa molecular weight antigen was characterized by immunopurification from NRK cells metabolically labeled with 35S-Met. This 32 kDa antigen appeared as part of a higher multimolecular membrane component of 300 kDa. A 170 kDa and a 70 kDa components were immunodetected in a semi-purified membrane fraction from rat liver, demonstrating that the antigen was a minor but very antigenic contaminant of the coated vesicle preparation used as immunogen. In conclusion, the labeled peri-Golgi structures may be part of the newly characterized trans-Golgi network and/or of the reticular/vesicular endosomal, prelysosomal structure recently described.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells

In a pilot clinical study carcinoma patients with malignant ascites or pleural exudates have been treated locally with autologous lymphocytes activated ex vivo and redirected towards tumour cells with bispecific monoclonal antibodies. BIS-1, the bispecific monoclonal antibody used in this study, combines specificity against a tumour-associated antigen, AMOC-31, present on carcinomas, with a specificity against the CD3 complex on T lymphocytes. Patients selected for treatment had malignant pleural or peritoneal effusions. Treatment consisted of isolating autologous peripheral blood lymphocytes, ex vivo activation, incubation with bispecific monoclonal antibodies and injection at the effusion site of these BIS-1-redirected lymphocytes. To evaluate the effects of the bispecific monoclonal antibody, five patients received treatments with activated lymphocytes without bispecific antibodies. Effusion samples taken before and at various times after treatment were analysed by immunocytology and for the presence of the soluble factors carcinoembryonic antigen (CEA), interleukin-6 (IL-6), tumour necrosis factor (TNF), C-reactive protein and soluble CD8. In this way both immune activation and anti-tumour activity could be monitored. Conjugate formation between tumour cells and activated lymphocytes was seen as soon as 4 h after injection of BIS-1-redirected activated lymphocytes, followed by a disappearance or reduction of tumour cells after 24–48 h. In parallel with this, the soluble tumour marker CEA decreased in the effusion fluid following injection with the BIS-1-redirected lymphocytes. Furthermore, a steep increase in local granulocyte numbers was observed in the effusion fluid, which reached a maximum 24–48 h after the start of the treatment. Also levels of IL-6 and TNF were greatly elevated. The data suggest tha the treatment induces both antitumour activity and a strong local inflammatory reaction. This is accompanied by no or only minor local and systemic toxicity, i.e. mild fever, which disappeared as the local inflammatory reaction diminished 48–72 h after treatment.     

Parallels of genomic organization and of endogenous retrovirus organization in cat and man

A combination of technical advances (most notably heterologous cell fusion, high resolution G-banding, and molecular cloning) has contributed to an accelerated advance in genetic analysis in mammals. The present human genetic map contains over 400 gene assignments and the map is growing rapidly as each new molecular clone or immunological reagent is developed. In our laboratory, we have developed a panel of rodent X human somatic cell hybrids that have been utilized in chromosome assignment of several classes of genes including oncogenes (ras, raf) and endogenous human retroviral sequences (ERVL, 2, etc). Using similar techniques, a biochemical genetic map of the domestic cat has been derived. The cat has 19 chromosome pairs and, to date, 40 genes have been mapped to 16 linkage or syntenic groups. Comparison of linkage relationships between homologous enzymes has revealed a striking conversation of chromosomal linkage association between cat and man. A comparison of syntenically homologous, highly extended high resoultion G-banded chromosomes between the two mammalian families revealed that 20–25%, by length, of the human karyotype can be precisely aligned (chromomere to chromomere) between cats and man despite the evolutionary divergence of the species nearly 80 million years ago. Moderately repetitive families of retrovirus-related DNAs exist within the feline and the human genomes. We have isolated molecular clones of several members of the feline RD-114 retrovirus family from a genomic library of normal cat cellular DNA. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. Several sequences were apparently deleted, relative to the previously characterized inducible RD-114 genome. The env regions of a number of endogenous RD-114 sequences examined were substantially deleted or diverged; a subset of these sequences contained information at the position of the env region that was not homologous to inducible RD-114. The RD-114 virogenes were dispersed to several cat chrosomes that were localized using a panel of rodent x cat somatic cell hybrids. A comparison of the genetic properties of endogenous human retroviral sequences revealed several similarities between the human and feline status of endogenous retroviruses.     

Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of lectins

Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha^R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent Pha^R clones. These clones also exhibit (a) a decreased ability to bind [¹²⁵I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind¹²⁵I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A^R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Genetic characterization of methotrexate-resistant chinese hamster ovary cells

Summary In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 × 10⁻⁹ mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Classes I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This research was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada and the National Institutes of Health of the United States. W. F. was a Postdoctoral Fellow of the Medical Research Council of Canada.     

Isolation and Characterization of a Near-Haploid Human Cell Line

Mammalian somatic cells are usually diploid. Occasional rare human tumors have been shown to have a hypodiploid karyotype. We have isolated a near-haploid subclone (P1-55) from a heterogeneous human leukemia cell line, KBM-7. These near-haploid cells have approximately half the human diploid DNA content and have a haploid karyotype except for a disomy of chromosome 8 (25, XY, +8, Ph(+)). This cell line maintains a majority of cells with a near-haploid karyotype for at least 12 weeks in culture. By serial subcloning, we have isolated near-haploid subclones that maintain ploidy for at least 8 months in culture. Near-haploid cells can also be efficiently isolated from mixed ploidy cultures by size selection. The availability of this human near-haploid cell line should facilitate the genetic analysis of cultured human cells.