Cytogenetic methodologies for gene mapping and comparative analyses in mammalian cell culture systems

7s , .-

j

1987, Gene Anal Techn 4:75-85 .z__ -

Cytogenetic Methodologies for Gene Mapping and Comparative Analyses in Mammalian Cell Culture Systems

f

paper presents a comprehensive description of our methodology and includes techniques for establishing cell lines and somatic cell hybrids, for obtaining chromosomal preparations from different cell lines, for differentially staining chromosomes, and for in situ chromosome hybridization.

E s t a b l i s h m e n t o f Cell C u l t u r e s WILLIAM S. MODI,

Peripheral Lymphocytes

WILLIAM G. NASH,

For chromosomal analyses, short-term lymphocyte cultures have been established from human, orangutan, and domestic cat blood samples. The procedure for human and orangutan preparations differ from one another only with respect to the mitogen used, and both species yield excellent results. Satisfactory chromosomal preparations are only occasionally obtained from cat lymphocyte cultures. Three milliliters of heparinized whole blood are mixed with 40 ml RPMI 1640 medium (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Gibco) and 1.5 ml phytohemagglutinin (final concentration of 7.5 p.g/ml) (PHA; Burroughs Wellcome) in the human blood, or 1.6 ml pokeweed mitogen (Gibco) plus 2.0 ml of stock phorbol ester solution (final concentration of 10 p,g/ml) (see Appendix) in the case of the orangutan blood [18]. The culture mixture is placed in a tightly capped, horizontally positioned 150 cm 2 tissue-culture flask and incubated at 37°C for 96 hr.

A N N A C. FERRARI, and S T E P H E N J. O'BRIEN

Presented here are the detailed methods employed in our laboratory for gene mapping and cytogenetic analyses in human beings, in the domestic cat, and in other mammalian species. Included in the procedures are: 1) establishment of primary fibroblast and lymphoid cell cultures; 2) heterologous cell fusion for production of rapidly proliferating cell hybrids; 3) cellular transformation of primary fibroblasts using an oncogenic retrovirus; 4) cell synchronization for high-resolution banding of prometaphase chromosomes; 5) chromosome-banding procedures, including G-banding, alkaline G-11, and Q-banding; and 6) in situ hybridization of radiolabeled molecular clones to metaphase chromosomes for regional gene localization.

Over the past several years, cytogenetic procedures have been utilized in our laboratory for gene localization in both human and domestic cat cellculture systems [1-16]. Similar data have been reported for other species, and this information forms the basis for comparative gene mapping in mammals (9,12,17). Recently, we have also become interested in comparative chromosomal analyses with a primary focus on phylogenetic reconstruction among different members of the carnivore order. The methods we employ include modifications of standard procedures that have been adapted successfully in our laboratory. This From the Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland (W.S.M., W.G.N., A.C.E, S.J.O.) and the Department of Medicine, the Union Memorial Hospital, 201 E. University Parkway, Baltimore, Maryland (A.C.E). Present address of W.G.N. is H & W Cytogenetic Services, Inc., Sterling, VA 22170. Address reprint requests to: William S. Modi, Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, MD 21701-1013. Received February 9, 1987.

Primary Fibroblasts Tissue biopsies (0.2-1.0 g of skin, lung, kidney, heart, testes) are aseptically collected and rinsed in several washes of Hanks' balanced saline solution (HBSS). Tissue should be digested and planted within a few hours of collection; however, when delay is necessary (for example, when material is shipped to our laboratory) the tissue piece is immersed in 5 ml of MEM Alpha (MEMA) tissueculture medium (Gibco) containing penicillin (100 units/ml), streptomycin (10 ~g/ml), and amphotericin B (fungizone) (2.5 ~g/ml; Gibco), supplemented with 10% inactivated fetal bovine serum and shipped on wet ice (4°C). For culturing, the tissue is minced with a curved scissors in HBSS and treated in one of three manners [19] described below.

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/87/$03.50

."

-_.,,J l'~

76 1987, G e n e A n a l T e c h n 4 : 7 5 - 8 5

~ J

. .___-

Method 1. Washed, minced tissue is placed in 10-15 ml of MEMA containing penicillin, streptomycin, amphotericin B, trypsin (0.25%; Gibco), and collagenase CLS II (5 mg/ml, about 600 units/ ml; C o o p e r B i o m e d i c a l ) in a sterile 125-ml Wheaton bottle containing a stir bar. The sample is stirred at 37°C for 30 min, after which the fluid is removed and centrifuged (150 g, 6 min), and the cell pellet is resuspended in 20 ml of MEMA containing 10% serum. Additional MEMA plus penicillin, streptomycin, amphotericin B, trypsin, and collagenase is added to the remaining sample, and the process is repeated several times until tissue pieces are no longer visible. The first fraction collected often contains tissue surface contaminants in addition to cells and may be discarded if ample tissue is available. The remaining supernatant fractions are pooled, and the cells are seeded in 5 ml of MEMA containing 10% serum, antibiotics, and fungizone in a 25-cm 2 tissue-culture flask. Cell growth is observed and the culture passaged when confluent. Method 2. Washed, minced tissue pieces are suspended in 1-5 ml of MEMA containing antibiotics, fungizone, trypsin, and collagenase. The contents are incubated at 37°C for 30 min or at 4°C for 6-10 hr followed by mechanical disaggregation through pipetting. The mixture is centrifuged and the cell pellet plated out as described above. Method 3. Washed, minced tissue pieces are plated out directly in a 25-cm 2 tissue-culture flask without enzymatic or mechanical disaggregation. We have found that the first of these procedures usually yields the greatest number of viable cells, although it is the most time consuming. Variations of these general protocols may be found elsewhere [19]. Finally, if disaggregation cannot be carried out immediately following sampling, minced tissue pieces may be stored by cryogenetically freezing in liquid nitrogen in MEMA containing 10% serum, antibiotics, fungizone, and 10% dimethyl sulfoxide (DMSO). Somatic Cell Hybrid Panel Construction Hybrids are derived by polyethylene glycol (PEG) mediated fusion of fresh human or feline leukocytes to rodent fibroblast cells (mouse RAG or Chinese hamster E36). The rodent cells lack the hypoxanthine phosphoribosyl transferase (HPRT) gene, and this allows the selection of hybrids in

hypoxanthine/aminopterin/thymidine (HAT) medium [20,21]. Approximately 5 x 10 6 rodent and 1 x 10 7 human or cat cells are mixed in 0.2 ml of a 50% PEG solution (M.W. = 1450; American Type Culture Collection) in Ham's F-12M medium (Gibco) (without serum). Triplicates of the above mixture are made, and each, along with a control tube from each parent, is incubated for a different time interval between 5 and 12 min at 37°C. The fusion mixture is then centrifuged (300 g, 6 min), the supernatant aspirated, the cells washed with HBSS, resuspended in F-12M medium containing 10% serum, plated in a 100-mm tissue-culture dish, and allowed to grow until confluent (24-48 hr). In an effort to establish single cell hybrid clones, cells are subcultured by splitting 1:20 and seeded at a density of 2 x 10 3 cells per 100-mm tissue-culture dish on selective medium: H a m ' s F-12M containing 10% serum and HAT supplement ( I 0 - 4 M hypoxanthine, 4 x 10 - 7 M aminopterin, 1.6 x 10 -5 M thymidine). Hybrid cells will adhere to the flask during growth. Parental rodent cells ( H P R T - ) are eliminated by the selective medium; parental human or cat lymphoid (floating) cells (HPRT ÷) are eliminated by the passage of only adherent cells. For positive and negative controls, parent cells are plated in selective medium (rodent cells should die) and complete medium (both parental cell types should grow normally). Two to four weeks later colonies 1-2 mm in diameter are visually selected, then removed by trypsin digestion using porcelain cloning cylinders (Fisher). Approximately 100 colonies per fusion are chosen and each is expanded as a separate hybrid clone. In order to establish a hybrid panel useful for gene mapping in human beings and cats, the following procedures were followed. The procedures used for both human beings and cats are identical. A single vial of each of the 100 hybrid clones was expanded to three confluent T-150 flasks, the cells were harvested, and an isozyme extract was prepared by sonication in 0.05M Tris (pH 7.1), 1.0 mM Na-EDTA. The extracts were electrophoretically typed for 3 0 - 5 0 isozyme structural genes of known chromosomal location in human beings. Rodent x human hybrids retain the entire rodent chromosomal complement, but lose human chromosomes in different combinations. The details of interpreting the isozyme scores are discussed elsewhere [15]. Hybrids that retain no human isozymes or all of the human iso-

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

1987, G e n e A n a l Techn 4 : 7 5 - 8 5

zymes were discarded as they are of little use in chromosome mapping. From the others, hybrids with few isozyme markers but with a strong isozyme reaction indicating a high frequency of that chromosome's retention were selected. To these were added as many hybrids as were required to have at least four hybrids positive for each human chromosome. Then the extent of discordance in the panel of each marker with all other chromosome markers was maximized by adding hybrids that achieved this goal. At this stage, we generally had 50-70 hybrid candidates selected for our panel. Because of the known instability of unselected chromosomes in heterologous hybrids, especially through a freeze cycle, each candidate hybrid was expanded in six T-150 flasks, then the cells from these flasks were pooled and divided into 20 aliquots that were cryogenically frozen. This step, although laborious, was useful because the extensive genetic characterization of a now frozen aliquot could be repeated in the future with 19 sister vials from precisely the same original freeze lot without a freeze and thaw between them. Thus, we were ready to thaw one vial for each of our panel members and expand it in tissue culture in order to perform isozyme typing, chromosomal analysis, and batch DNA and R N A extraction from the same passage of cells. Once the Gbanded chromosomal analysis was completed, an alignment of isozyme and chromosome scores could be cross-checked. The chromosomal analysis showed heterogeneity between hybrid clones in both the combination of human chromosomes and the number of cells that had a particular chromosome. Because of this heterogeneity, it is necessary to analyze 20-50 metaphase spreads for each hybrid in order to estimate chromosome frequencies quantitatively. Finally, a fraction of hybrids exhibited extensive rearrangement of human chromosomes, especially when Chinese hamster (E36) cells were the rodent parent. Such hybrids were excluded from the hybrid panels. Our present working human-mapping panel has 44 hybrids [5,6], and our cat-mapping panel has 41 hybrids [3,22].

Metaphase

Chromosome

Preparation

Peripheral Lymphocytes Cells (human and orangutan) are synchronized by adding 5 - b r o m o d e o x y u r i d i n e ( B r d U ; Sigma)

(20-100 ~g/ml) 72 hr after culture initiation [231. Seventeen hours later, cells are washed twice with HBSS, resuspended in fresh medium (20% serum and appropriate mitogen) containing thymidine (10 .5 M) and allowed to grow an additional 5.5-6 hr. Colcemid (Gibco 0.07 ~g/ml) is added 20 rain before harvest. At harvest, the culture solution is transferred to four 15-ml centrifuge tubes, and the cells are centrifuged (200 g, 10 min). The supernatant is carefully removed with a pipette such that the mononuclear layer remains undisturbed, the cells are resuspended in 10 ml 0.075 M KCI, and incubated at 37°C for 30 min. Cells are again centrifuged, pooled into one 15-ml tube, vigorously resuspended using a desk-top vortex unit in 10 ml Carnoy's fixative, and placed at 4°C overnight. After overnight fixation, cells are washed three times in 6 - 8 ml of fresh fixative and resuspended in 2 0 - 3 0 volumes (usually 3 - 6 ml) of fresh fixative. One or two drops of this cell suspension are dropped onto wet slides that are allowed to dry horizontally for 1-2 rain on a rack suspended 3-10 cm above a 65°C water bath. As a means of preservation, cells may be stored in fixative at - 7 0 0 C for up to one year before making slides, and slides may be stored at 4°C for up to six months. Because of the photosensitivity of BrdU, all cells and slides should be handled in subdued light.

Primary Fibroblasts We have successfully established cell lines containing fibroblasts from a number of mammalian species. Growth rates of some cell lines are reasonably high and thus are suitable for chromosomal analysis only during the first 2 - 8 passages; subsequent growth rates are considerably reduced. Other lines grow so slowly that chromosome harvest even at early passages is limited. In an effort to overcome low mitotic indexes, we have successfully stimulated cellular proliferation in cell lines from several carnivore (felid, canid, ursid) species by infecting cells with feline sarcoma virus (FeSV). This procedure causes morphologic transformation and rapid cell growth without inducing chromosomal rearrangements. A mink (Mustela vison) lung fibroblast cell line (ST/aut) that is productively infected with both feline leukemia virus (FeLV) and the Synder-Thelein strain of FeSV is used as a source of virus. The supernatant from subconfluent ST/aut cells is collected, filtered (0.20 ~m Nalgene filter), and di-

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

~ r'~

•

..--- . j

78 1987, Gene Anal Techn 4:75-85

.:" J

% jPP V

i ,

\

i.

|

El

C

U m2

1t F2

m n

U °1 YX

Figure 1. a. Primary fibroblast cell line from the domestic cat. l ~ t e flat, adherent cellular morphology, b. Cell line shown in (a) following transformation with feline sarcoma virus (FeSV) after three weeks. Note refractile, loosely adherent properties of cells, c. G-banded karyotype of the cheetah, Acinonyx jubatus, d. C-banded metaphase cell of the leopard cat, Prionailurus bengalensis.

luted 1:3 with fresh medium and added to target cells. Polybrene (Aldrich) is added to the virus mixture at a final concentration of 2 - 8 Cg/ml. Cells are incubated and then refed and passaged

o

d when confluent. After 1-2 weeks (felid) or 6-10 weeks (canid, ursid),foci of transformed cells appear (Figure la and b). Metaphase cells are obtained by shaking and dislodging loosely adherent dividing cells from 4 - 6 subconfluent 150-cm 2 flasks [24] or by removing the entire cell population of 1-2 flasks using trypsin digestion (4 ml of 0.05% trypsin, 1 mM EDTA in HBSS per 150 cm 2 flask, incubated at 37°C for 2-10 min). Cells are centrifuged (150 g, 8 min), the pellet is resuspended in 10 ml of

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

1987, Gene Anal Techn 4:75-85

0.075 M KCI and incubated at 37°C for 20-30 rain. Cells are again centrifuged, the supernatant is decanted, and the cells are gradually fixed in 10 ml Carnoy's fixative and placed at 4°C for a time period ranging from 10 min to overnight. In order to increase mitotic indexes or decrease chromosomal condensation, Colcemid (0.07 ~g/ml, 4-12 hr before harvest) or ethidium bromide (5 v~g/ml, 2.5 hr before harvest; Sigma) [25] may be added, respectively. Following fixation, cells are washed three times in fresh fixative and finally resuspended in 20-30 cell volumes (usually 1-3 ml) of fixative. One or two drops of this suspension are then dropped onto wet slides and allowed to dry under elevated (50-70%) humidity conditions such that metaphase chromosomes will appear refractile with a minimum amount of cytoplasmic background w h e n observed under phase-contrast microscopy. Cells and slides may be stored as described for lymphocyte preparations.

Somatic Cell Hybrids Chromosomal preparations from somatic cell hybrids are obtained as described above for primary fibroblast cell lines except that FeSV transformation is unnecessary. Loosely adherent, dividing cells are easily obtained by the mitotic shakeoff procedure.

Differential Staining Throughout the past two decades a number of advances has been made regarding the development of differential chromosomal staining procedures. For example, Q-banding [26], G-banding [27], and R-banding [28] all produce longitudinal chromosomal differentiation enabling identification of homologous elements both within and between species. C-banding enables determination of the amount and location of constitutive heteroc h r o m a t i n [29], w h e r e a s a n u m b e r of fluorochromes, when used in conjunction with the appropriate counter stain, produce regional banding patterns or highlight specific heterochromatic regions [30]. Staining with a silver nitrate solution has been shown to identify the chromosomal locations of transcriptionally active 18S + 28S ribosomal genes (rDNA) [31]. Primate chromosomes have been shown to stain differentially against a rodent background in somatic cell hybrids using the alkaline Giemsa (G-11) staining procedure [32,33].

For G-trypsin banding, slides of metaphase chromosomes are aged by placing them in a 37°C incubator for 5-20 days followed by immersion in HBSS containing 0.025% trypsin for 30-200 sec at room temperature. Slides are then rinsed in dH20 before staining in 2% Giemsa in phosphate buffer for 2-6 min (Figure lc). In C-banding, slides are treated at room temperature in 0.2N HCI for 1 hr, rinsed, dried, and treated with saturated Ba(OH)2 at 50°C for 5-15 min. Slides are again rinsed, dried, covered with 2 x SSC (pH 7.0), and then incubated at 60°C in a 100% humidity chamber for 1 hr. Slides are dehydrated in an ethanol series (70%, 80%, 95%) before being stained in 2-4% Giemsa for 2-10 min (Figure ld). Silver staining for the nucleolus organizer region (Ag-NOR) is carried out by flooding slides with 50% silver nitrate (AgNO3) containing 0.05% formalin and incubating at 60°C for 1-6 hr followed by rinsing and drying (Figure 2a). Both human and domestic cat somatic cell hybrid panels are used in gene-mapping studies [1-16]. Human and cat elements are identified against a rodent background using G-11 and Gbanding procedures (Figures 2b to d, 3a). For G-11 staining, freshly prepared phosphate buffer is prewarmed to 37°C. Slides are hydrated by soaking in dH20 (37°C, 30 min) before being stained in a mixture of 47 ml of buffer and 3 ml of G-11 stain (37°C, 4-6 min), followed by rinsing and drying. Human or cat chromosomes stain light blue, whereas the rodent background elements stain much more darkly (Figure 2b and c). Slides in which chromosomes of the two different species are poorly differentiated may be destained in an alcohol series (70%, 95% ethanol, 100% methanol) and restained. This procedure is invaluable in the identification of interspecific translocations that are difficult to document using G-banding (Figure 2b). In Situ H y b r i d i z a t i o n

Radiolabeling We have utilized both nick translation [34] and random primer extension [35] to construct radioactively labeled hybridization probes.

Nick Translation The following (35 txl total) are added to an Eppendoff tube and incubated at 14°C for I hr: 7 txl (3H)deoxyodenosine 5'-triphosphate, tetrasodium salt

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

~ (~'~ /~(~

..--

j

80 1987, Gene Anal Techn 4:75-85

f

.,-

f

,

h

II

C

Figure 2. a.

Silver-stained metaphase cell of the mountain

l]-6"ff~ma concolor. Arrows indicate location of nucleolus organizer region (Ag-NOR) on chromosome pair El. b. G-1 lstained metaphase cell of human-hamster somatic cell hybrid. Lightly staining human chromosomes are encircled. Interspecific translocations are indicated by t. c. G-ll-stained metaphase cell of c a t - m o u s e somatic cell hybrid. Lightly staining cat chromosomes are encircled, d. G-banded rectaphase cell of human-hamster somatic cell hybrid. Human chromosomes are encircled and numbered.

(New England Nuclear NET-657, 40-60 Ci/mmol, approximately 24 ~,M), remove ethanol solvent by

drying in a vacuum; 7 ~1 (3H)-thymidine 5'-triphosphate, tetrasodium salt (NET-520A, 90-110 Ci/mmol, approximately 23 ~M); 4 ~1 (3H)-deoxycytidine 5 ' - t r i p h o s p h a t e , t e t r o s o d i u m salt (NET-601A, 40-60 Ci/mmol, approximately 48 ~M); 2 ~1 dGTP at 375 IxM (Sigma); 1 ixl DNA (plasmid containing insert at 0.5 p,g/ul); 3.5 ixl 10× Nick Translation Buffer [10]; 1.5 txl Pancreatic DNAse I at 0.1 ixg/ml (Bethesda Research Labs, BRL); 1 ~1 Escherichia coli DNA Polymerase I at 10,000 units/ml (BRL); and 15 ~1 H20.

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

1987, Gene Anal Techn 4:75-85

,/

tl

After incubation, the reaction is stopped by adding 5 ~1 of 20% SDS in 100 p,M EDTA. Five microliters of carrier DNA (sheared salmon sperm DNA at 2 mg/ml) are added, and the total volume is brought to 50 p,l by adding 5 p,l dH20. Specific activity of the probe and efficiency of nick translation are then determined by trichloroacetic (TCA) precipitation [36]. This method typically yields hybridization probes at 1-3 x 107 cpm per microgram DNA.

Figure 3. a. G-banded metaphase cell of cat-mouse somatic ce-"~[-h-~rid. Cat chromosomes are encircled and numbered. b. G-banded human peripheral lymphoid metaphase chromosomes after in situ hybridization with radiolabeled human endothelial cell growth factor (ECGF) cDNA clone 1 [14]. Arrow shows autoradiographic silver grain occurring on chromosome 5. c. Q-banded human metaphase chromosomes after in situ hybridization of radiolabeled Moloney leukemia virus integration site #2 cDNA clone (Anagnou et al., unpublished). Arrow shows autoradiographic grain on chromosome 5. d. Distribution of 257 autoradiographic grains observed along the chromosomes of 81 human metaphase cells after in situ hybridization with the human ECGF clone [14].

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

K

-

..__-

f ..-- _...

~J

82

1987, Gene Anal Techn 4:75-85

R a n d o m Primer Extension Probe DNA, 100-300 ng, (plasmid-containing insert) and 10 Ixl 5 x Random Primer Buffer [35] are diluted to a total volume of 30 ixl in dH20, boiled for 5 min and immediately put on ice. The following (50 Ixl total) are then added: 7 ixl (3H)deoxyodenosine 5'-triphosphate, tetrasodium salt (NET-657), remove ethanol solvent by drying; 5 ~1 (3H)-thymidine 5'-triphosphate, tetrasodium salt (NET-520A); 5 ~1 (3H)-deoxycytidine 5'-triphosphate, tetrasodium salt (NET-601A); 2 ~1 d G T P (375 ixM); 2 ~1 bovine serum albumin (Pentax fraction V) (I0 mg/ml) (BRL); 1 Ixl E. coli DNA polymerase I Klenow fragment (8,000 units/ ml) (BRL); and 5 Ixl dH20. The reaction mixture is allowed to stand at room temperature for 2-10 hr. Then, SDS/EDTA and carrier DNA are added, and TCA precipitation is performed as described above in the nick translation procedure. This method typically yields probes at 3 - 8 x 107 cpm per microgram DNA. Unincorporated label may be removed from labeled D N A following both radiolabeling procedures using either of two Sephadex (Pharmacia) separation methods [36] outlined below.

Vertical Column Method. A 10-ml plastic disposable pipette mounted vertically is packed with G-50 Sephadex that has been equilibrated with Sephadex Running Buffer. As the buffer flows through the column into a collection vessel, it is replaced at the top of the column. The sample (50-100 p~l) is loaded onto the top of the packed Sephadex, the buffer is continually replaced, 12 1-ml fractions are collected, and 5-~1 aliquots from each fraction are counted in a liquid scintillation counter. The fractions containing the first peak are pooled, the total volume is reduced to about 400 Ixl by extraction using secbutanol [36], and the DNA is precipitated by adding ammonium acetate to a final concentration of 2.0 M followed by the addition of two volumes of absolute ethanol at -20°C. After centrifugation, the supernatant is decanted, the pellet is resuspended in 100 Ixl of Tris-EDTA (TE), and a 5-1xl aliquot is counted in a liquid scintillation counter to determine concentration. The sample is stored at - 20oc. Spin Column Method. A 1-ml blue pipette tip is placed along with a collar into a 12 × 75 mm collection tube, and the tip is packed with equilibrated G-50 Sephadex. The tube is centrifuged at

600 g for 8 min, causing the buffer in the pipette tip to drain into the collection tube. The pipette tip is transferred to a clean collection tube, and up to 100 ~xl of sample is loaded on top of the packed Sephadex in the pipette tip. The tube is spun again (600 g, 10 min); most of the sample liquid volume should come through the column into the collection tube and contain the labeled DNA. Unincorporated label will remain in the column. A 5-1xl aliquot is counted to determine probe concentration and efficiency of recovery.

In Situ Hybridization Slides are treated [37] with pancreatic RNAse A (Sigma) (200 ixg/ml in 2 x SSC, pH = 7.0) by pipetting 50 p~l of solution onto each slide, covering slides with coverslips, and incubating them at 37°C for 1 hr in a 100% humidity chamber. Slides are washed in four changes of 2 x SSC (pH 7.0) 5 min each at room temperature, followed by dehydration in an ethanol series (70%, 80%, 90%, and 95%), all in Wheaton jars containing stir bars. Slides are denatured in a filtered (Whatman #1) 70% formamide solution in 2 x SSC (pH 7.0) at 70°C for 2 min in a plastic Coplin jar positioned in a water bath followed by dehydration in a cold (4°C) ethanol series. Labeled probe DNA is diluted in a hybridization solution (pH 7.0) containing 50% formamide, 10% dextran sulfate, 2X SSC, 20 mM sodium phosphate (pH 6.8), 10 x Denhardt's solution and carrier DNA (e.g., sheared salmon sperm DNA) at a concentration 500 times that of the probe. Several probe concentrations between 1 ng/ml and 50 ng/ml should be tried. Probe mixture is denatured by incubation at 70°C for 5-10 min immediately prior to hybridization and put on ice. Hybridization is carried out by placing 30 Ixl of hybridization solution on each slide, covering the slides with coverslips, and placing covered slides in a 100% humidity chamber at 37°C for 16 hr. Following hybridization, slides are washed for 5 min in each of five changes of 2 x SSC in 50% formamide, and five changes of 2 x SSC, all pH 7.0, and at 40°C. Slides are again dehydrated in an ethanol series at room temperature.

Autoradiography and Chromosome Identification Nuclear track emulsion (NTB-2; Kodak, handled in complete darkness) is incubated at 45°C for 1 hr prior to a 1:1 dilution (20 ml each) with distilled

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

84

1987, Gene Anal Techn 4:75-85

22. Berman, E. J., Nash, W. G., Seuanez, H. N., and O'Brien, S. J. (1986)Cytogenet. Cell Genet. 41,114-120. 23. Dutrillaux, B., and Viegas-P6quignot, E. (1981) Hum. Genet. 57, 93-95. 24. Wolff, S., and Perry, P. (1974) Chromosoma 48, 341-348. 25. Ikeuchi, T. (1984) Cytogenet. Cell Genet. 38, 56-61. 26. Caspersson, T., Zech, L., Johansson, C., and Modest, E. J. (1970) Chromosoma (Beri.) 30, 215-227. 27. Seabright, M. (1971) Lancet, II, 971-972. 28. Dutrillaux, B., and Lejeune, J. (1971) C.R. Acad. Sci. Paris Ser. D. 272, 2638-2640. 29. Arrighi, F. E., and Hsu, T. C. (1971) Cytogenetics 10, 81-86. Sumner, A. T. (1972) Exp. Cell Res. 75, 304-306. 30. Schweizer, D. (1981) Hum. Genet. 57, 1-14. 31. Goodpasture, C., and Bloom, S. E. (1975) Chromosoma 53, 37-50. 32. Bobrow, M., and Cross, J. (1974) Nature 251, 77-79. 33. Burgerhout, W. (1975) Humangenetik 29, 229-233. 34. Rigby, P. W. J., Dieckmann, M., Rhodes, C., Berg, P. (1977) J. Mol. Biol. 113,237-251. 35. Feinberg, A. P., and Vogelstein, B (1983) Anal. Biochem. 132, 6-13. Feinberg, A. P., and Vogelstein. B. (1984) Anal. Biochem. 137, 266-267. 36. Maniatis, T., Fritsch, E. E, and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, New York. 37. Harper, M. E., and Saunders, G. (1981) Chromosoma (Bed.) 83,431-439. 38. Perry, P., and Wolff, S. (1974) Nature 251, 156-158. 39. Chandler, M. E., and Yunis, J. J. (1978) Cytogenet. Cell Genet. 22, 352-356.

AppendixmBuffers and Solutions Stock Phorbol Ester Solution Dissolve 5 mg phorbol 12-myristate 13-acetatel-4O-methyl ester (Sigma) in 10 drops of dimethyl sulfoxide and add to 25 ml of complete RPMI 1640 medium; solution is then filtered (0.2 ~m), aliquoted, and stored at -20°C.

Carnoy' s Fixative

g/l), pH to 7.0 with 10 N sodium hydroxide (NaOH).

G-11 Stain Grind 1 g of Giemsa stain powder (Fisher) in a few milliliters of warm glycerol in a mortar and pestle; add additional glycerol to a total volume of 66 ml and stir at 60°C overnight. Cool the solution, add 66 ml of absolute ethanol, and stir at room temperature I - 2 hr. Store in a dark bottle.

G-11 Buffer Use 0.05 M dibasic sodium phosphate (Na2HPO4) (7.1 g/l), pH to 11.3, with 10 N NaOH added immediately before using.

Nick Translation Buffer (10 × ) Mix 0.5 M Tris base; pH to 7.2 with hydrochloric acid (HC1), 0.1 M magnesium sulfate (MgSO4), 1 mM dithiothreitol (DTT), and 500 ~g/ml bovine serum albumin (BSA Pentax Fraction V).

Random Primer Extension Buffer (5 × ) Solution 0:1.25 M Tris-HCl, 0.125 M MgCI 2, pH to 8.0, and store at 4°C; solution A: 1 ml of solution 0 and 18 p.l of [3-mercaptoethanol, and store at 4°C; solution B: 2 M Hepes titrated to pH 6.6 with 4 N NaOH, and store at 4°C; solution C: hexadeoxyribonucleotides (IBI) evenly suspended in TE at 90 optical density units/milliliter, and store at -20°C. Mix solutions A:B:C in a ratio of 100:250:100 to make 5 x buffer and store at - 20°C.

Denhardt' s Solution (100 x )

Add 3 volumes absolute methanol to 1 volume glacial acetic acid.

Mix Ficoll, 10 g; polyvinylpyrrolidone, 10 g; BSA (Pentax fraction V), 10 g; and dH20 to 500 ml; then filter, aliquot, and store at -20°C.

Phosphate Giemsa buffer (pH 6.8)

Macllvane' s Buffer (pH 5.5)

Add 1 volume 0.01 M sodium phosphate monobasic (NaH2PO4 • H20) (1.38 g/l) to 1 volume 0.01 M sodium phsophate dibasic (NazHPO4) (1.42 g/l).

A d d 65 ml o f 0.1 M a n h y d r o u s citric acid (H3C6HsO7) (19.2 g/l) to 85 ml of 0.2 M sodium phosphate dibasic (Na2HPO4) (28.4 g/l).

2 x SSC

Wright Stain

Add 0.3 M sodium chloride (NaCI) (17.53 g/l) to 0.03 M sodium citrate (Na3C6HsO7 • 2H20) (8.82

Stir 1.25 g Wright stain (EM Sciences or MC/B) in 500 ml of absolute methanol and stir at 37°C until

© 1987ElsevierSciencePubfishingCo., Inc., 52 VanderbiltAve., New York, NY 10017

1987, Gene Anal Techn 4:75-85

f ~ ------___

water in a 50-ml conical centrifuge tube. Hybridized slides are immersed in the diluted emulsion for 1-2 sec, vertically placed in a rack, and dried at room temperature for 1-2 hr. Dried slides are placed in a slide box containing a desiccant (Drierite). The boxes are sealed with labeling tape and placed in a second light-proof container. Slides are stored at 4°C for an exposure period ranging from 7 to 60 days. After the appropriate exposure time (which is estimated by observing approximately three chrom o s o m a l grains per cell on a d e v e l o p e d and stained test slide), remaining slides are similarly developed in filtered, diluted (1:1 with dHzO) either Dektol (2 min), or D-19 (4 min) photographic developer (Kodak), followed by rinsing in distilled water (20 sec) and fixation (Kodak fixer, 5 min), all at 15°C. Slides may now be exposed to light and are washed in dHzO for 5 min followed by air drying. Metaphase chromosomes may be differentially stained using several procedures. If cells had been substituted with BrdU, then high-resolution G-bands are obtained as follows (Figure 3a). Slides are flooded with Hoechst 33258 (1 ixg/ml; Sigma) in 2 x SSC (pH 7.0) for 15 min in the dark, followed by rinsing and drying. Slides are covered with 2 x SSC (pH 7.0) and illuminated with long-wave ultraviolet light for 1 hr [38]. Slides are rinsed, dried, and finally stained in a solution of Wright stain: phosphate buffer (1:3) for 4 - 8 min at room temperature [39]. If bands are not sharp, they may often be improved by destaining in three solutions, 95% ethanol (2 min), 0.5% HCI in 95% ethanol (30 sec), and absolute methanol (2 min) followed by restaining with Wright stain [39]; however, destaining often removes autoradiographic silver grains. G-bands of variable quality may be obtained with this Wright stain procedure on unsynchronized cells. Fluorescent Q-bands may be prepared by dehydrating slides for 1 hr in 95% ethanol prior to staining with quinacrine mustard (50 Ixg/ml; Sigma) in Macllvane's buffer (pH 5.5) for 10-80 min followed by rinsing in two washes of buffer, drying, mounting in fresh buffer or in a saturated sucrose solution, and visualization under ultraviolet light with Zeiss filter set 09 (Figure 3c).

Analysis of In Situ Hybrids In order to identify the chromosomal site where a particular clone maps, it is necessary to determine the distribution of grains along the entire karyo-

type by examining 50-100 cells. By plotting a histogram showing the frequency of grains observed along each chromosomal arm, an elevated signal at one chromosomal location relative to nonspecific background hybridization at other c h r o m o s o m a l sites should b e c o m e apparent (Figure 3d).

References 1. O'Brien, S. J. (1976) Proc. Natl. Acad. Sci. USA 73, 4618-4622. 2. Lemons, R. J., O'Brien, S. J., and Sherr, C. J. (1978) Cytogenet. Cell Genet. 22, 255-259. 3. O'Brien, S. J., and Nash, W. G. (1982) Science 216, 257-265. 4. Nash, W. G., and O'Brien, S. J. (1982) Proc. Natl. Acad. Sci. USA 79, 6631-6635. 5. O'Brien, S. J., Bonner, T. I., Cohen, M., O'Connell, C., and Nash, W. G. (1983) Nature 303, 74-77. 6. O'Brien, S. J., Nash, W. G., Goodman, J. L., Lowy, D. R., and Chang, E. H. (1983) Nature 302,839-842. 7. Reeves, R. H., Nash, W. G., and O'Brien, S. J. (1985) J. Virol. 56, 303-306. 8. Seigel, L. J., Nash, W. G., Harper, M., Wong-Staal, E, Gallo, R. C., and O'Brien, S. J. (1984) Science 223, 175-178. 9. O'Brien, S. J., Seuanez, H. N., and Womack, J. E. (1985) in Maclntyre, R. J. (Ed.): Molecular Evolutionary Genetics (Monographs in Evolutionary Biology Series) (MacIntyre, R. J., ed.), pp. 519-589, Plenum Press, New York. 10. Nienhuis, A. W., Bunn, H. E, Turner, P. H., Gopal, T. V., Nash, W. G., O'Brien, S. J., and Sherr, C. J. (1985) Cell 42,421-428. 11. Hentze, M. W., Keim, S., Papadopoulos, P., O'Brien, S. J., Modi, W. S., Drysdate, J., Leonard, W. J., Harford, J. B., and Klausner, R. D. (1986). Proc. Natl. Acad. Sci. USA 83, 7226-7230. 12. O'Brien, S. J. (1986) in Proceedings of Genetic Engineering of Animals: An Agricultural Perspective (Evans, J. W., and Hollaender, A., eds.), pp. 139-149, Plenum Press, New York. 13. O'Brien, S. J. (1986) Molecular genetics in the domestic cat and its relatives. Trends Genet. 2, 137-142. 14. Jaye, M., Howk, R., Burgess, W., Ricca, G. A., Chiu, I-M., Ravera, M., O'Brien, S. J., Modi, W. S., Maciag, T., and Drohan, W. H. (1986) Science 233,541-544. 15. O'Brien, S. J., Simonson, J. M., and Eichelberger, M. A. (1982) In Techniques in Somatic Cell Genetics, (Shay, J. W., ed.), pp. 513-524, Plenum Press, New York. 16. Brownell, E., Kozak, C. A., Fowle, J. R., III, Modi, W. S., Rice, N. R., and O'Brien, S. J. (1986) Am. J. Hum. Genet. 39, 194-202. 17. Lalley, P., McKusick, V. A. (in press) Cytogenet. Cell Genet. 18. Wiley, J. E., and Meisner, E (1984) In Vitro 20, 932-936. Ryder, O. A., personal communication. 19. Freshney, R. I. (1983) Culture of Animal Cells. A Manual of Basic Technique. Alan R. Liss, Inc., New York. 20. Pontecorvo, G. (1975) Somatic Cell Genet. 1,397-400. 21. Davidson, R. 1., and Gerald, P. S. (1975) Somatic Cell Genet. 2, 165-176.

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

-

.._--- -~ ~--~3

1987, G e n e Anal Techn 4 : 7 5 - 8 5

dissolved ( I - 2 hr), then fdter through Whatman # 1 paper and store in a dark bottle.

Sephadex Running Buffer Mix 10 mM Tris-HCl (pH 8.0), I mM disodium ethylene diamine tetraacetate. 2HeO (EDTA), 100 mM NaC1, and 0.1% N-lauroylsarcosine (Sigma).

Wright Stain Buffer (pH 6.8) Add 1 volume 0.06 M sodium phosphate dibasic (NaeHPO4) (8.52 g/l) and 1 v o l u m e 0.06 M (KHePO 4) (8.16 g/l).

Tris-EDTA (TE), pH 7.4 Mix 10 mM Tris-HC1 (pH 7.4) and I mM EDTA (pH 8.0).

© 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017