Specialized Technique Lauren E. Bertonazzi I. Lymphocyte Preparation 1.

Procedure for separating lymphocytes from plasma and erythrocytes (1) Flotation on a combination of Ficoll and Sodium Metrizonate (pp. 467-468 Protocol 27.1) (2) After centrifugation, lymphocytes will be concentrated at the interface between the Ficoll-Hypaque and the plasma, granulocytes in the pellet (3) Suitable for A. PHA stimulation B. Chromosome analysis C. Enriching viable cells D. First step in purification of lymphocyte subclasses 2. Procedure for Blast Transformation (1) Stimulate with mitogens to quantify the immunocompetence of the cells. (p. 468 Protocol 27.2) (2) May be used to produce mitosis for chromosomal analysis of peripheral blood (pp. 262-263 Protocol 16.7) II. Autoradiography 1. Macroautoradiography (1) Used to localize material in blots from electrophoresis (2) Suitable isotopes (p. 469 Table 27.1) A. Low energy emitters (A) Gives high intracellular resolution when used with a thin emulsion B. Intermediate energy emitters (A) Gives localization at the cellular level C. High energy emitters (A) Gives poor resolution at microscopic level but used for chromatograms and blots from DNA, RNA, and protein electrophoresis 2. Microautoradiography (1) Used to localize material in microscope preparations (2) After incubation with appropriate isotopically labeled precursor and exposure to emulsion, silver grains can be seen in areas where radioisotope was incorporated (p. 469 Protocol 27.3) (3) Useful tool for determining the distribution of isotope incorporation within a population, but less useful for quantitation III. Time-Lapse Recording 1. Developed primarily to show naturally slow processes at an accelerated rate 2. Microscopy technique is critical link in the whole process (pp. 473-474 Protocol 27.4) 3. Imagining software can be used to measure position, area, and intensities of image (1) Useful in automatic analysis of cell behavior 4. Fluorescence images (1) Useful for evaluating cell translocation, cell spreading, intensity of fluorescence, and/or morphological characteristics IV. Cell Synchrony

1. Used to follow the progression of cells through the cell cycle (1) Cell population is blocked metabolically or fractionated so they are all at the same phase and progress in the cycle 2. Cell Separation (1) Sedimentation at unit gravity (2) Centrifugal elutriation A. Preferable if a large number of cells is required (3) Fluorescence-activated cell sorting A. Used in conjunction with a nontoxic DNA stain B. Lower yield than for centrifugal elutriation (4) Mitotic shake-off A. Monolayer cells tend to round up at metaphase and detach when the flask is shaken 3. Blockade (1) DNA synthesis inhibition A. Agents to inhibit S phase are toxic and may lead to nonviable cells, cells that have been blocked but are viable, and unblocked cells (A) Thymidine (B) Hydroxyurea (C) Cytosine arabinoside (D) Aminopterin (2) Nutritional deprivation A. Serum is removed from the medium for 24 h and then restored, where the cycle is resumed in synchrony B. Does not work well in transformed cells V. Culture of Amniocytes 1. Coverslip method (pp. 479-480 Protocol 27.5B) 2. Closed-Tube Culture System (pp. 476-479 Protocol 27.5A) (1) Advantages of robustness, simplicity, and minimal use of resources 2. Diagnostic capabilities (1) Inborn errors of metabolism (2) Sex-linked conditions (3) Autosomal dominant or recessive conditions (4) Detection of trisomies and sex chromosome aneuploidy VI. Culture of Cells from Poikilotherms 1. Dissociation technique (1) Use proteolytic enzymes as a chelating agent, and fetal bovine serum or modified media formulation 2. Fish cells (1) Cell lines derived from early-stage embryos (pp. 481-482 Protocol 27.6A) A. Cells are pluripotent B. ZEF fibroblast cell line used as a feeder layer for primary cultures (2) Primary cultures derived from early-stage embryos (p. 483 Protocol 27.6B) A. Maintain a diploid chromosome number when derived on a feeder layer of ZEF fibroblasts (3) Cell lines derived from late-stage embryos (pp. 483-484 Protocol 27.6C) A. ZF29, ZF13, and ZF4 fibroblastic cell lines

3. Insect cells (1) Use of baculovirus for gene cloning (pp. 484-485 Protocol 27.7) A. Grown in Sf9 cells from the fall armyworm VII. In Situ Molecular Hybridization 1. Molecular techniques (1) Lysate analysis A. Southern blot and PCR methods B. Tissue biopsies are homogenized and a loss of information on heterogeneity and small subpopulations occurs C. Quantitation is simpler and more accurate (2) RNA In situ hybridization (pp. 485-488 Method 27.8) A. Allows for the visualization of gene expression in cells within their histological context B. Based on specific binding of labeled nucleic acid probe to sequence in a tissue sample, followed by visualization of the probe C. Appropriate for cells from culture and tissue from samples of whole or biopsied organs as success depends on retention of nucleic acid sequences in the sample 2. Fluorescence in situ hybridization in the analysis of genes and chromosomes (pp. 489-490 Method 27.9) (1) Most direct method of determining the linear order of genes on chromosomes and analyzing numerical and structural variations in individual cells (2) Applications include diagnosis of genetic disease and identification of gene deletions, translocations, and amplifications in cancerous tissue (3) Advantages of this method are A. Nonisotopic B. Rapid C. Good for data storage and manipulation D. Sensitive VIII. Somatic Cell Fusion 1. Cell hybridization technique (pp. 491-492 Protocol 27.10) (1) Somatic cells fuse when cultured with inactivated Sendai virus or with polyethylene glycol (2) Some cells will progress to nuclear fusion and may progress through mitosis so both sets of chromosomes replicate together. May be interspecific fusion (3) Some cell lines may undergo spontaneous fusion 2. Selection of hybrid clones (1) Method of selection depends on the species of origin of the parental cell lines, the growth properties of the cell lines, and whether selectable genetic markers are present (2) The HAT system may be used to isolate hybrids made between pairs of mutant cell lines deficient in certain enzymes 3. Nuclear transfer (1) Nuclei can be isolated by centrifuging cytochalasin B-treated cells and fusing the extracted nuclei to recipient whole cells or enucleated cytoplasts in the presence of polyethylene glycol (2) Primary useful application is in cloning individual animals IX. Production of Monoclonal Antibodies (pp. 493-495 Protocol 27.11)

1. Hybridomas (1) Produced by fusing a nonsecreting myeloma cell with an antibody producing Blymphocyte in the presence of polyethylene glycol (2) Can be cultured indefinitely and will produce unlimited quantities of antibody 2. Screening (1) Hybridoma culture supernatant should be screened early for desired reactivity patterns (2) Use of ELISA for reactivity to the immunogen 3. Subcloning (1) Serial dilution of cells or sorting with an automated cell deposition unit may be done (2) Cells will be cultured and screened for positive hybridomas which are expanded and frozen X. DNA Transfer 1. Study function of individual genes and regulatory sequences (1) Cloned DNA is often maintained as a bacterial plasmid A. Can attain a high copy number during bacterial growth B. DNA is purified from bacteria before use (2) Specific sequences can be studied via subclones containing desired sequence A. Sequences can be further manipulated by linking to a reporter gene whose products can be assayed 2. Coprecipitation with calcium phosphate (1) Exogenous DNA is mixed with calcium chloride and is added to a solution containing phosphate ions (2) Calcium phosphate DNA coprecipitate is formed which is readily taken up by mammalian cells in culture so the exogenous DNA will be expressed 3. Lipofection (pp. 497-498 Protocol 27.12) (1) Method is based on an ionic interaction of DNA and liposomes to form a complex which can deliver functional DNA into cultured cells (2) Advantages include A. Higher efficiency B. Ability to transfect a wide variety of eukaryotic cell lines C. Relatively low cellular toxicity D. Basic procedure can be adapted for transfection with other molecules 4. Electroporation (pp. 498-499 Protocol 27.13) (1) A high cell concentration is briefly exposed to a high voltage electric field in the presence of the DNA to be transfected (2) Small holes generated in the cell membrane allows DNA to enter the cell and become incorporated into the genome (3) Performed at a constant capacitance setting (4) Linear relationship between DNA concentration, DNA uptake, and reporter gene expression (5) Suspension cells more easily transfected by electroporation because they do not have to be detached from the vessel 5. Retroviral infection (1) High efficiency of gene transfer (2) Able to incorporate larger DNA fragments than plasmids (3) Infect host cells spontaneously

(4) Introduced gene becomes permanent (5) Process of insertion is not harmful to the host cell and does not cause any other genetic alteration 6. Yeast artificial chromosomes (1) Provide a genome that is capable of packaging larger sequences of DNA than bacterial plasmids (2) Downstream posttranslational processing such as found in eukaryotic cells (3) High yield, easy to maintain, and stable culture system 7. Mammalian artificial chromosomes (1) Principles of yeast artificial chromosomes may be applied to mammalian systems (2) Enables incorporation of large mammalian sequences containing one or more structural and regulatory genes into one construct (3) Introduced into mammalian cells by monochromosomal transfer techniques