[19 Chromosome Sorting by Flow Cytometry

By Marty Bartholdi, Julie Meyne, Kevin Albright, Mary Luedemann, Evelyn Campbell, Douglas Chritton, Larry L. Deaven, and L. Scott Cram The 24 human chromosome types can be sorted by flow cytometry with 90 purity in quantities sufficient for chromosome specific DNA library construction1 or direct hybridization on filters.2 The techniques that need to be brought together to sort one million chromosomes of a single type, or 50 thousand of each type, on a commercially available flow cytometer are...

[20 Methods for Chromosome Banding of Human and Experimental Tumors in Vitro

Thompson Chromosomal analysis is increasingly becoming a necessity in studies of somatic cell genetics. The major emphasis of this chapter will be to describe several common cytogenetic procedures which can be utilized to identify the chromosomal constitution of human and experimental tumors grown in vitro. Emphasis has been placed on discussion of methods for banding analysis of established cell lines, although a brief description of procedures for analysis of...

Exotoxin A

Microbial toxins are potent biological agents that are often capable of killing mammalian cells by enzymatically inactivating normal cellular function. If it were possible to harness the activities of toxins and redirect them to selectively kill or damage discrete populations of cells, powerful reagents might become available for use both as tools for cell biology and cell genetics and as novel drugs for the treatment of cancer or other diseases. Toward this end Pseudomonas exotoxin A (PE) was...

Table I

Mutant or Variant Forms of S49 Cells Deoxycytidine kinase deficiency 23, 24 Thymidine kinase deficiency 25 Uridine-cytidine kinase deficiency 26 Orotate phosphoribosyltransferase-OMP decarboxylase deficiency. 25, 27 Orotate phosphoriboxyltransferase-OMP decarboxylase, elevated levels. 25, 27 Hypoxanthine-guanine phosphoribosyltransferase substrate affinity alter- 28 ation Purine-nucleoside phosphorylase deficiency. 29 Ribonucleotide reductase alterations with abnormal responsiveness to dGTP 29...

Nuclear Hybrids

The use of enucleation procedures results in the production of nuclear and cytoplasmic parts termed karyoplasts and cytoplasts, respectively.1 By fusing a cytoplast with a karyoplast, a viable reconstituted cell can be produced (Fig. 1). Alternatively, by fusing a cytoplast from one cell to another whole cell, a cytoplasmic hybrid or cybrid is formed (Fig. 1). Finally, by fusing a karyoplast to another whole cell it is possible to form a nuclear hybrid (Fig. 1). The various protocols for the...

[16 Principles of Electrofusion and Electropermeabilization

Urnovitz Biological membranes exhibit limited permeability to both electrolytes and nonelectrolytes. Throughout evolution, selective transport systems have been developed to achieve and control uptake and release of specific solutes charged or uncharged, low and high molecular weight. Increasing membrane permeability is of significant interest for biotech-nological applications with noted emphasis on modification of cellular Copyright 1987 by Academic Press,...

[18 Toxin Antitoxin Selection for Isolating Heterokaryons and Cell Hybrids

Most studies of the behavior of cell hybrids have employed genetic selection techniques, such as the HAT system,1 to isolate cell fusion products. Although a number of different systems based on this general approach have been extremely useful, there are several practical and theoretical problems associated with their use. For example, only special cell lines that lack the required enzymes can be used. Although in some cases this problem can be overcome by mutagenizing and selecting for cells...

Contributors to Volume 151

Article numbers are in parentheses following the names of contributor. Affiliations listed are current. Shin-ichi Akiyama (4), Department of Cancer Chemotherapy, Institute of Cancer Research, Faculty of Medicine, Kago-shima University, 1208-1 Usuki-cho, Ka-goshima 890, Japan Kevin Albright (19), Experimental Pathology Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Marty Bartholdi (19), Experimental Pathology Group, Los Alamos National Laboratory, Los Alamos, New Mexico...

[22 Gene Mapping with Sorted Chromosomes By Roger V Lebo and Barry D Bruce

Sorted chromosomal DNAs isolated by single laser chromosome sorting and analyzed by standard restriction enzyme analysis were used to map the -, y-, and -globin1 and insulin genes to the short arm of chromosome 11. Hybridization of radiolabeled probe to chromosomes sorted onto spots with a single laser sorter determined the Y-chromosome containing histogram peak2 and further sublocalized the myc gene.3 Spot-blot analysis of chromosomes separated with a high-resolution dual-laser chromosome...

Info

Antitoxin protection against toxin killing. The relative survival in each aliquot was estimated from the number of human fibroblast cells remaining attached 2 days after an overnight toxin treatment for (A) antiricin toxin-injected cells and (B) antiabrin-injected cells. inject cells with a constant amount of ricin A chain (e.g., 15 ng ml) using different osmotic lysis conditions (see below), then wash away the free ricin A chain and put the cells back in culture. After 3 hr the cells...

[23 Transfer and Selective Retention of Single Specific Human Chromosomes via Microcell Mediated Chromosome Transfer

Stanbridge Human gene mapping has been greatly facilitated by the development of interspecific somatic cell hybrids, in which human cells are fused to rodent cells, resulting in human-rodent hybrids.1 These hybrids initially contain a complete set of chromosomes from both parents, but then rapidly segregate out most of their human chromosome complement in a 1 V. A. McKusick and F. H. Ruddle, Science 196, 390 (1977). Copyright 1987 by Academic Press, Inc. METHODS IN...

[2 Cultured S49 Mouse T Lymphoma Cells

By Theodoor van Daalen Wetters and Philip Coffino S49 is a mouse T cell lymphoma that has been adapted to grow in culture. It has characteristics that are of general utility to investigators who make use of cultured cells. The cells grow quickly, with a generation time of approximately 16 hr.1 They have a stable near-euploid karyotype.2 They grow in stirred or stationary suspension culture. The cells do not adhere to the culture vessel and associate only loosely with each other. This obviates...

[3 Liver Cell Lines By G J Darlington

A variety of liver cell lines have been derived from tumorgenic and nontumorgenic hepatocytes and have been adapted to growth as permanent cell lines. Only a subset of those described in the literature will be presented here in great detail. Those which have been examined for only a small number of hepatospecific phenotypes or have not received wide distribution have not been covered within the scope of this chapter. While the culture and maintenance of normal diploid hepatocytes as a...

[1 Chinese Hamster Ovary Cells

Chinese hamster ovary CHO cells have been extensively used for genetic analysis in tissue culture since the pioneering work of Puck, who first isolated this cell line.1 These cells have been used for the isolation of mutants affecting intermediary metabolism DNA, RNA, and protein synthesis membrane functions and several more complex forms of cell behavior such as cell growth and endocytosis. A recent compilation of CHO mutants lists more than 80 classes of mutants isolated using this cell...