Cytogenetic Methods for the Mouse: Preparation of

Chromosomes, Karyotyping, and in Situ Hybridization

The ability to examine the chromosomes of murine cells is rapidly becoming essential to the
mouse molecular geneticist. Although these technologies exist, the protocols are scattered
throughout the literature, and in some cases different protocols exist for the same procedure.
We attempt here both to augment and to consolidate this body of work. This review provides a
complete set of procedures for manipulating mouse chromosomes. Prospective sources of
metaphase chromosomes are discussed and detailed protocols are outlined for their preparation
and characterization. We assume that the reader has little or no cytogenetic background and
have included a troubleshooting guide to assist the process. Protocols for in situ hybridization to
metaphase spreads that allow the rapid localization of sequences using nonradioactive probes
are also included. Variations in the protocols described here abound and alternatives are
indicated.

Within the past decade the laboratory mouse has emerged as a model system for studying the
details of gene expression and regulation in higher eukaryotes. Interest in the mouse is fueled by
the availability of a large number of mutations affecting not only physical characteristics of the
animal but also molecular processes controlling the expression of specific genes (1). The ability to
generate transgenic animals, e.g., microinjection of DNA into the fertilized egg (2-4), infection of
embryos with retroviral vectors (5-7), or the manipulation of pluripotent embryonic stem (EQ2
cells (8,9), has also allowed molecular geneticists to introduce and study the expression of foreign
genes integrated into the mouse genome. A number of these integration events have disrupted
endogenous mouse genes (10-17). In some cases the disrupted gene is known, i.e., a previously
characterized mutation for the gene exists (10,12,13,17). Many of the insertional events, however,
occur in genes not previously identified (11,14-16; F. D. Costantini, unpublished observations; E. J.
Robertson, unpublished observations). More recently, technical advances using homologous
recombination in ES cells to target genes are generating null alleles of cloned genes for which no
mutations have been identified (18-21).

These genetic manipulations require that the integrity of the genome be maintained and, as a
result, molecular biologists are now required to double as cytogeneticists. Using cytogenetic
methods, biologists can assess the karyotype of a cell as well as localize endogenous sequences.
Alternatively, part of the initial characterization of a transgenic mouse line carrying inserted DNA
that disrupts an endogenous mouse gene should be the localization of the integration event, i.e.,
the chromosome and the position on that chromosome at which the DNA integrated. In addition,
tissue culture cells are particularly subject to chromosomal abnormalities, and subsets of
aneuploid cells will be selected over a period of continuous culture (22). As a result, it would be
unwise to spend months cutting and splicing DNA, transfecting and selecting for homologous
recombination events in ES cells, and attempting to make chimeric mice without knowing if the
cells possess a normal karyotype. The experimental strategies outlined here simplify cytogenetic
manipulations and provide the necessary background for identifying specific chromosomes.

SOURCE OF TISSUES/CELLS
Metaphase chromosomes are derived from either embryonic stem cell cultures or tissue taken
from the animal proper. The selection of a source of chromosomes is determined by several
considerations. For example, if the chromosome preparation is to be made from tissue excised
from the animal, can the animal be sacrificed? The karyotype of an animal may need to be known
before a breeding program can be instigated, necessitating that the animal be alive after the
procedure. Sources of metaphase chromosomes are limited to those tissues containing a fraction
of cells that are actively undergoing cell division. From post partum mice this restricts the choice
of starting tissue to the hemopoietic lineages of the bone marrow and circulating blood and to
primary cell cultures derived from tissue biopsies.

Bone marrow contains populations of stem cells and committed progenitor cells that continually
undergo cell division, supplying the peripheral blood with both red blood cells and nucleated
leukocytes (23). Such preparations generally contain a small number of metaphase spreads and
the procedures can be performed without tissue culture facilities. Results are usually obtained in 1
or 2 days.

Lymphocytes of circulating blood retain their ability to divide only when cultured in the presence
of a mitogen such as concanavalin A (24). Since a 3- to 4-day culture period is required, the blood
sample must be collected and handled in a sterile fashion. Although more complex, these
preparations generally contain a greater percentage of cells in metaphase. Because a nominal
quantity of blood is required to initiate the culture, the procedure can also be readily used to
genotype adult mice without having to sacrifice the animal. Unfortunately, while mitogen-
stimulated human blood cultures divide rapidly (25), mouse blood cultures may not respond in the
same way (26), dividing too slowly or not at all. Preparations that divide, however, offer a
renewable source of metaphase chromosomes from the same animal.

The unpredictability of blood preparations necessitates the availability of an additional source of

mitotitally active cells. A more reliable supply is obtained through tissue biopsy coupled with
primary cell culture. Tissue biopsies, including the tip of the tail and ear clippings, can be grown
successfully in culture for a limited period of time with no chromosome abnormalities (27).
Primary cell cultures can be started from a small amount of tissue, can generate large numbers of
metaphase spreads, and can be made with tissue obtained from neonatal mice as well as from
adults. While both sources of cells divide well in primary culture, neonatally derived cells divide
much more rapidly and reliably.

PREPARATION OF METAPHASE CHROMOSOMES

The procedures in this section are divided into four major categories on the basis of the starting
tissue or cell type. The materials and solutions needed within this section are listed separately.
Materials and reagents are sold by a number of companies and in most cases may be substituted
for one another.

Materials and Solutions

Plastic ware and other disposables are purchased presterilized and when possible in bulk form.
These include, 3-ml syringes, 25-gauge and 26-gauge needles, conical centrifuge tubes, T-25 tissue
culture flasks, and tissue culture dishes (3, 6, and 10 cm). The protocols described below require a
table-top clinical centrifuge capable of spinning tubes at 500g. The solutions are listed roughly in
the order of their appearance in the text. DMEM (Dulbecco’s modified Eagle’s medium) can be
purchased from GIBCO Laboratories. Hypotonic KC1 is 0.56% KC1 prepared in distilled water and
should be stored at room temperature. Trypsin-EDTA (1X; 0.25% trypsin, ImM EDTA) can be
purchased from GIBCO. Once thawed, a stock bottle should be stored at 4°C for no more than a
month. The colcemid solution (100X; GIBCO) is 2 pg/ml prepared in sterile water. Carnoy’s fixative
is methanol and glacial acetic acid (3:l; prepared just before use). Complete medium is DMEM, 2
mM glutamine (from GIBCO as a 100X stock [200 mM]), 10% fetal bovine serum (HyClone
Laboratories), penicillin:streptomycin (from GIBCO as a 100X stock [lO,OOO U:lO,OOO pg]), and
100 pM ,&mercaptoethanol. A 10 PM stock solution of methotrexate (Sigma, under the name
amethopterin) is prepared in DMEM and stored at -20°C. A 1 mM stock solution of thymidine
(Sigma) is prepared in DMEM and stored at -20°C. Supplemented blood culturing medium is RPM1
1640 (GIBCO), 25 mM Hepes, 2 mM glutamine, 0.1 mg/ ml gentamycin sulfate (Schering), 20%
fetal bovine serum, 50 pg/ml concanavalin A (Sigma), and 50 pg/ml heparin (Sigma). Prepare this
medium on the day of use. PBS, 1X, is Dulbecco’s phosphate-buffered saline without calcium
chloride and magnesium chloride. This can be purchased from GIBCO as either a sterile 1X stock or
a 10X stock that is diluted with sterile water.

Tissue Culture Cells

The protocols presented here assume a working knowledge of ES cells and tissue culture in
general. A number of books and manuals provide the information necessary to deal with these
cells (9,22,27). We commonly grow cells in 6-cm dishes, finding that the number of cells derived
from a dish of this size is sufficient for the preparation of many slides (210). It is, however,
important not to allow the cell cultures to overgrow. As cultures approach confluence, cells
become mitotically arrested and will produce few metaphase spreads. Our experience is that cells
harvested 2 days before or after passage are rapidly dividing and are in sufficient number for
preparing chromosomes (9).

Cells should be fed (5 ml of complete medium) 3-4 h prior to the addition of colcemid; this ensures
that the culture is growing at the optimal rate. Add 50 ~1 of 100X colcemid to the 6-cm dish
containing the cells. Incubate the dish at 37°C for 45-75 min. Following this incubation, wash the
cells once with 1X PBS, trypsinize, collect in complete medium, and then transfer to a 15-ml
conical centrifuge tube. Collect the cells in the clinical centrifuge (500g) for 5 min. Aspirate the
supernatant and loosen the cell pellet before proceeding to the next step. Slowly resuspend the
cells in 5 ml of warmed (37’C) hypotonic KC1 (shaking to ensure a single-cell suspension) and
incubate at room temperature for 10 min. Following this incubation, centrifuge at 500g for 5 min.
Decant the supernatant and vortex briefly to resuspend the cell pellet. While vortexing lightly, add
a few drops of Carnoy’s fixative. Note: Once a few drops have been added, stop vortexing! The
cells now become fragile and are subject to lysis. Since it is important to prevent clumps of cells
from forming, continue to shake the tube by hand, adding a total of 5-7 ml of fixative. Finally, mix
thoroughly by inverting the tube. Incubate the cells, without shaking, at room temperature for 15
min. Centrifuge the cells at 500g for 5 min, decant the supernatant, and loosen the cell pellet.
Repeat the fixation step twice more. The three incubations in fixative ensure complete fixation
and dehydration. After the final incubation, cells can be used to prepare metaphase chromosomes
(see below). Experience has shown that the best results are obtained when the chromosomes are
prepared the same day; however, if the cells are to be stored for later use, resuspend them in 5-7
ml of fixative and store at -20°C. Cells fixed in this manner will keep for weeks.

Bone Marrow

Recovery of bone marrow is a difficult and messy task, which is in part due to the small size of an
adult mouse. The bones of the hind legs offer the only reasonable site from which marrow can be
readily recovered.

Sacrifice a mouse by cervical dislocation. Dissect out the bones of the back legs by cutting the legs
free at the hip joint. It is important, however, not to damage the ends of the femurs. The most
convenient technique is to break the pelvis with dissection scissors on either side of the backbone.
Invert each leg (including the attached muscle), pulling it free of skin. Sever the feet from the legs
at the ankle, being careful not to damage the ends of the tibias. Trim away the bulk of the leg
muscles with dissection scissors. Snap the trimmed legs in half at the knee joint by hyperextension
and strip away the remaining muscle of each femur and tibia with tissue paper. Cut away the
extreme ends of each bone, turning the bone into an open-ended tube. Fill a 3-ml syringe with
DMEM warmed to 37°C and fit the syringe with either a 25- or 26-gauge needle, depending on the
size of the smallest hole of a given bone. Flush the marrow out into a small tube. The marrow from
a single mouse (two femurs and two tibias) should be collected in ~2 ml of medium. Using a
Pasteur pipet, disrupt the cells of the marrow to a fine colloid. The cells at this point will not lyse
when pipetted up and down.

Direct bone marrow preparation. In a 15-ml conical centrifuge tube, add 500 ~1 of the bone
marrow suspension to 9 ml of hypotonic KC1 (warmed to 37’C), 1 ml 1X trypsin-EDTA, and 100 ~1
of 100X colcemid. Mix and incubate at 37’C for 30 min. Following this incubation, sediment the
cells at 500g for 5 min. Decant the supernatant and vortex briefly to disrupt the cell pellet. While
vortexing lightly, add a few drops of Carnoy’s fixative and, once a few drops have been added,
stop vortexing! Since it is important to prevent clumps of cells from forming, continue to shake the
tube by hand, adding a total of 5-7 ml of fixative. Finally, mix thoroughly by inverting the tube. The
remainder of the fixation process is performed as described under Tissue Culture Cells. Note that
bone marrow contains copious amounts of structural proteins that denature in the presence of
the fixative, forming a white precipitous material; this material does not affect the chromosome
preparation.

Methotrexate-synchronized bone marrow culture (Indirect bone marrow preparation). An

unfortunate drawback to bone marrow preparations is that they generate few metaphase
chromosome spreads; at any moment, the percentage of cells in metaphase is small. The fraction
of cells in metaphase can be increased, however, by briefly incubating the marrow in serum-
containing medium and an inhibitor of DNA synthesis. Methotrexate, which blocks thymidine
synthesis, is effective. The “synchronized” culture is allowed to proceed to M phase by washing
the cells free of inhibitor and incubating them in the presence of thymidine. Because bone marrow
can be cultured in a sealed flask, a tissue culture incubator is not necessary.

In a T-25 tissue culture flask, add 1 ml of bone marrow suspension to 9 ml of supplemented blood
culture medium without concanavalin A and heparin. Screw the flask closed and incubate the
marrow at 37% for 3-6 h. After incubation add 100 ~1 of 10 PM methotrexate (10e7 M final
concentration) and incubate at 37°C overnight (15 h). Transfer the cells to a 15-ml conical
centrifuge tube and spin in the clinical centrifuge (5OOg) for 5 min. Decant the supernatant and
wash the marrow twice by resuspending the cells in 10 ml of complete medium at 37°C. Each time,
centrifuge the cells for 5 min at 500g. Resuspend the final cell pellet in 5 ml of supplemented
blood culture medium without concanavalin A and heparin. Add 50 ~1 of 1 mM thymidine (lo-’ M
final concentration) and incubate the cells at 37°C for 2.5-3 h. The incubation is performed in a
closed centrifuge tube laid horizontally in the incubator. Add colcemid (50 ~1 of the 100X stock) to
the cultured cells and incubate at 37°C for an additional 30 min. Sediment the marrow (5OOg, 5
min) and remove the supernatant by decanting. Loosen the pellet and resuspend the cells by
slowly adding 9 ml of hypotonic KC1 at 37°C. Shake the tube constantly while adding the KC1 to
prevent clumping. At this point the marrow cells will not lyse if vortexed lightly. Incubate the cells
at 37°C for 30 min and fix as described above.

Concanavalin A-Stimulated Peripheral Blood Cell Cultures

This technique involves the collection and culture of peripheral blood lymphocytes. The
lymphocytes can be stimulated to divide by the addition of such mitogens as concanavalin A,
phytohemaglutinin, or lipopolysaccaride to the culture medium (24-26). A full discussion of this
protocol can be found in Davisson and Akeson (26).

Dispense 4-ml aliquots of supplemented blood culture medium into 5-ml sterile tubes (prepare
two tubes per mouse). To collect the blood sample, lightly anesthetize the animal with the
anesthetic Avertin [see Hogan et al. (3) for details of its correct administration and use]. Place the
anesthetized mouse under a warm lamp for a few minutes (about 5) to effect dilation of the blood
vessels. The most convenient method for blood collection is to nick the tail vein close to the distal
end of the tail or to remove the tip of the tail completely. Clean the tail with 70% ethanol and cut
using a sterile scalpel blade. Collect 4 or 5 drops of blood directly into each culture tube, taking
care not to touch the tube with the tail. Cap the tube and mix immediately by inverting the tube.

Culture the tubes for 2 days at 37°C in either an airheated incubator or a shaking water bath (-30
rotations/min). If a stationary incubator is used, the tubes should be shaken manually two to three
times per day. The cultures should be a dark cherry red and show no evidence of red blood cell
aggregation. If the cultures quickly change to brownish yellow or clumps appear, the preparation
is probably contaminated and should be discarded. After the 2-day culture period, add 4 ~1 of
100X colcemid directly to the culture tubes, mix well, and culture for an additional l-2 h. Centrifuge
the tubes (5OOg, 5 min) and carefully aspirate the supernatant. Suspend the pellets from each
tube in 5 ml of hypotonic KC1 and incubate for 10 min at room temperature. It is important to
ensure that the pellets are suspended as single cell suspensions. This procedure causes the red
blood cells to lyse and also causes the lymphocytes to swell. Sediment the lymphocytes at 500g for
5 min. Aspirate the supernatants carefully, making sure to leave the uppermost opaque layers,
composed of the lymphocytes, undisturbed. Gently tap the tubes to ensure that the cell pellets are
well dispersed and slowly add fresh Carnoy’s fixative, while gently flicking each tube. Add a total of
5 ml of fixative to each tube and incubate for 15 min at room temperature. The remainder of the
fixation process is performed as described under Tissue Culture Cells.

Primary Fibroblast Cultures Derived from Tissue Biopsies

The most difficult aspect of cultures from biopsies is the maintenance of sterility. After tissue
fragments are recovered, all subsequent manipulations are performed in a tissue culture hood
using only sterilized instruments. Cultures are readily obtained from tip of the tail or ear tissue
biopsies (27). Wipe the tail or ear of the animal with a cotton swab soaked in 70% ethanol. Using
sharp sterile scissors cut off a small fragment of tissue and transfer to a sterile 6-cm petri dish,
adding a small volume (~0.5 ml) of culture medium. Using the scissors, mince the tissue into very
small fragments (<l mm’). Transfer the tissue fragments, together with the culture medium, into a
3-cm tissue culture dish with a Pasteur pipet. Immobilize the tissue fragments by trapping them
under a sterile coverslip. To sterilize coverslips, soak them in 70% ethanol, pick up each one
individually using a pair of fine forceps, drain off the excess alcohol, and touch them to a flame to
burn off residual alcohol. Incubate the cultures, undisturbed, for 3-4 days to allow the cells to
attach to the substrate and proliferate. After this time, fibroblasts should have migrated from the
tissue fragments and be readily discernible as a halo of cells surrounding the tissue. Gently remove
the coverslip using sterile forceps, taking care not to dislodge the fibroblasts. Depending on the
relative numbers of fibroblasts obtained, this primary culture can be used directly to prepare
metaphase spreads. Alternatively, the cultures can be expanded using routine tissue culture
protocols. Briefly, wash the cells with 1X PBS and trypsinize to dislodge the cells from the culture
dish. Add complete medium to neutralize the trypsin and vigorously pipet the resulting suspension
so that the cells are well dispersed; transfer the suspension into a fresh 3-cm dish. Grow the cells
until the culture becomes semiconfluent. Add colcemid directly to the culture (final concentration
1X), incubating for an additional 60 min. Following this incubation, cells are treated as described
under Tissue Culture Cells for embryonic stem cell cultures.

PREPARATION OF GIEMSA-BANDED CHROMOSOMES

Materials and Solutions

Use standard microscope slides, preferably with a frosted end. The slide preparation protocols
below require a slide warmer or a thermal adjustable hot plate. The availability of a phase contrast
inverted microscope is beneficial but not an absolute requirement. A humidifying chamber may be
necessary when the local atmosphere is low in humidity. This chamber can be constructed from a
10-cm tissue culture dish. The floor of the dish should be covered with filter paper saturated with
water, and slides are incubated inside, propped against a pipet tip to give them a slight downward
angle.

The required reagents are listed roughly in the order of their appearance in the text. Trypsin-EDTA
(10X; 0.5% trypsin, 5.3 mM EDTA) can be purchased from GIBCO. Once thawed, a stock bottle is
stored at 4°C for no more than a month. KPB is 25 mM potassium phosphate buffer, pH 6.8 (1.76 g
of potassium dibasic phosphate and 2.02 g of potassium monobasic phosphate per liter). Store at
4°C.

Giemsa stock solution can be prepared from 1 g of Giemsa powder (Fisher) dissolved in 66 ml of
glycerol. Incubate this solution, with constant stirring, at 56°C for 2-3 h. After the solution cools to
room temperature, add 66 ml of methanol. Store at 4°C for 2 weeks or longer before use. The
“aged” stock is stored at 4°C. Giemsa staining solution is prepared as a mix of 29 ml of KPB, 10 ml
of methanol, and 1 ml of Giemsa stock solution. This stain should be prepared prior to use and
may be retained at room temperature during the day. Weak dipping buffer is prepared as a mix of
5 ml of 1X Trypsin-EDTA and 45 ml of KPB. Strong dipping buffer is prepared as a mix of 2.5 ml of
10X Trypsin-EDTA and 47.5 ml of KPB. Each should be prepared fresh on the day of use. SSC, IX, is
0.15 M sodium chloride and 15 mM sodium citrate at pH 7.5.

Slide Preparation

Preliminary considerations. (a) When starting with fixed cells stored at -2O”C, sediment the cells (8
min, 500g) and resuspend in fresh fixative prior to use. Incubate at room temperature for 15 min.
Centrifuge the cells and suspend in fresh fixative.

(b) The cell suspension from which chromosomes are prepared should be homogeneous, i.e.,
without clumps, and have the turbidity of a 0.3 A, bacterial culture.

(c) The slides must be cleaned before chromosome spreads can be made. Normally, it is sufficient
to take slides directly from the box, wiping them clean with 70% ethanol and tissue paper. While
somewhat unorthodox, this is convenient because slides can be prepared as needed.

Metaphase chromosomes are prepared by air-drying fixed cells onto a glass microscope slide. The
spreading of the cell suspension and subsequent evaporation of fixative cause the cell membranes
to rupture and release the chromosomes.

Using a Pasteur pipet, take up 100-300 ~1 of a homogeneous cell suspension. Hold a cleaned slide
by the frosted end at a slight downward angle and release 2 drops of cell suspension side by side
from a height of 3-5 cm. If the relative humidity of the lab is 30% or higher, the usual case, simply
wait a few moments to allow the slide to dry. If, however, the relative humidity is low, the
preparations.will dry too quickly and the chromosomes will not spread well. To eliminate the
problem, immediately place the slide in a humidifying chamber, again waiting a few moments for
the slide to dry. Under a phase contrast inverted microscope, check the slide for the presence of
metaphase spreads. Figure 1 presents the inverted microscopic view of a freshly prepared slide.
Swollen unlysed nuclei usually predominate in these preparations; superimposed on this
background are cell debris and metaphase chromosome spreads. In addition to looking for
metaphase chromosomes, examine the preparation for chromosome spreading as well as
size/shape (extension). The degree of spreading will vary from one set of chromosomes to
another. Consequently, a judgment as to whether the chromosomes are spread properly is based
on an average of several spreads. Chromosome spreading is ideal when the individual
chromosomes appear clustered, yet non-overlapping. Chromosomes displaying flawless extension
appear as cylindrical objects which are somewhat refractile (arrowheads, Fig. 1). Both parameters,
spreading and extension, result from the complicated interaction of several physical and
environmental factors. Experience has shown that varying specific factors can dramatically alter
the observed results. Table 1 provides a troubleshooting guide for the preparation of metaphase
chromosomes. It is a compilation of the major problems that commonly arise during the
preparation and staining of metaphase chromosomes.

Incubate slides that have passed these initial criteria on a slide warmer at 60°C for 2-3 h.
Thereafter place the slides in a box together with a small amount of desiccant and incubate
overnight at 37°C. This process is known as “aging the slides.” An alternative procedure is to place
freshly prepared slides in a box with desiccant and incubate at 37°C for 2-3 days, thereby
eliminating the need for a slide warmer. Aged slides are stored in a sealed box with fresh desiccant
at 4°C. The best results for subsequent procedures are obtained with recently aged slides.
However, using this storage method, we have been able to perform Giemsa banding and in situ
hybridization of chromosomes weeks later.

Giemsa Staining

Slides can be stained with Giemsa immediately after aging; however, slides stored at 4°C must first
be warmed to room temperature while remaining sealed in the storage box. This prevents
condensation and the accompanying destruction of chromosome structure.

The banding process is the result of two independent events: removal of chromosomal proteins by
trypsin and staining by Giemsa. Different laboratories have remarkably different opinions on the
correct method of achieving this goal and, as a result, two distinct banding protocols are provided.
In both, a number of factors, including the absolute age of the slide and the exact manner in which
the slide was stored, determine the stringency of the trypsin digestion. The troubleshooting guide
(Table 1) provides insights to many of the more common problems associated with the banding
process.

G-Banding No. 1 (9,28): Preliminary considerations. (a) Equilibrate a staining dish containing 2X
SSC in a water bath at 60°C.

(b) Freshly prepare weak dipping buffer (50 ml) and Giemsa staining solution as described under
Materials and Solutions.

(c) Standard microscope slides serendipitously fit into 50-ml screw-top disposable centrifuge
tubes, which can replace standard staining jars. Equilibrate a 50-ml centrifuge tube containing
weak dipping buffer on ice (4°C). Arrange seven additional 50-ml tubes in a rack at room
temperature. Four of the tubes should contain 45 ml of distilled water and the remaining three
tubes 45 ml of KPB each.

This banding strategy involves the partial denaturation of the chromosome spreads followed by
digestion with trypsin. Initially incubate the slides in 2~ SSC at 60°C for 60 min. The slides should
then be washed in three changes of distilled water, 2 min each, and stored under water until use.
The slides should be trypsinized and stained individually. The conditions of trypsin digestion are
such that the reaction is easily controlled. Incubate a slide in the weak dipping buffer at 4°C for 3-4
min. The dipping time of aged slides varies depending on the individual slide, its age, and manner
of storage. Properly stored slides usually require less than 3 min; however, the older they are, the
more rigorous the trypsin treatment must be. Slides stored at room temperature or in humid
environments will require rigorous trypsin digestion obtained by, for example, increasing the
temperature of the digestion. In general, storage in humid places should be avoided. The
appropriate dipping time is determined empirically for each batch of slides, judging by the quality
of the Giemsa banding pattern. Free the slide of excess trypsin with two washes in 1X KPB. Dip the
slide several times in each wash, and place face-up on the bench top. Pat dry the frosted end of
the slide with tissue paper. This prevents the staining solution from extending to this region and
picking up the oils left by fingers; oil will coat the slide and interfere with chromosome staining.
Flood the slide with Giemsa staining solution and stain for approximately 8 min. This period can
also be varied to strengthen or weaken the chromosomal banding pattern. Following incubation,
drain off the stain and dip the slide successively several times in 1X KPB and water. Air-dry the
slide before microscopic examination. Locate Giemsabanded chromosomes and photograph
suitable spreads (60X oil immersion). Chromosome spreads may be protected at this point with a
mounting medium and coverslip unless other procedures are to be performed, e.g., in situ
hybridization. In the latter case, the slides are quickly dipped in xylene to remove the immersion
oil and are sealed in a box with desiccant at 4°C for storage. These slides have a half-life in storage
of a few weeks.

G-Banding No. 2 (29): Preliminary considerations. (a) Prepare weak dipping buffer (50 ml), strong
dipping buffer (50 ml), and Giemsa staining solution as described under Materials and Solutions.

(b) Arrange five centrifuge tubes consecutively in a rack. The tubes contain weak dipping buffer,
strong dipping buffer, 70% ethanol, distilled water, and distilled water, respectively.

In this procedure, slides are treated with trypsin without a denaturation step. This method is
somewhat less consistent and more difficult to control but is much faster and less complicated
than G-banding No. 1. Trypsin digestion includes dipping the slide in the appropriate buffer,
stopping the reaction with ethanol, and rehydrating the slide in preparation for Giemsa staining.
Aged slides are dipped in either weak or strong dipping buffer depending on the individual slide,
its age, and the manner of its storage. Properly stored slides usually require dipping in weak
buffer, but the older they are, the more rigorous the trypsin treatment must be. Slides stored at
room temperature or in humid environments require incubation in strong dipping buffer. The
amount of time a slide is left in dipping buffer is also variable and can be accomplished in as little
as 1 s or as long as 1 min. The procedure is flexible, with the appropriate dipping buffer and time
being determined empirically for each batch of slides. Once a slide is trypsinized, it should be
immediately dipped in 70% ethanol; dip 2 or 3 times for a few seconds each. Dip the slide several
times in water and place face-up on the bench top. Pat dry the frosted end of the slide with tissue
paper and flood the slide with Giemsa staining solution. Stain the chromosomes for approximately
3 min; this time can be varied to strengthen or weaken the chromosomal banding pattern. Drain
off the stain and dip the slide several times in the remaining centrifuge tube containing water. Air-
dry before microscopic examination. Poststaining treatment of the slides is as described above.

KARYOTYPING MOUSE CHROMOSOMES

Chromosomal identification is based on morphological criteria (e.g., relative size) as well as the
defined banding pattern each chromosome exhibits through chemical or enzymatic reactions
(1,30). Although numerous banding protocols exist, the focus here is on the most commonly used
and recognized of these reactions, the G-banding or Giemsa staining of chromosomes. This section
is intended to provide a pragmatic approach to the identification of mouse chromosomes.
Emphasis is placed on the recognition of a “normal” karyotype and the identification of specific
chromosomes on the basis of known examples.

General Characteristics of a Metaphase Spread

An obvious, yet informative, first step is to count the number of chromosomes in a given spread.
Since the haploid mouse genome contains 19 autosomes and a sex chromosome, metaphase
spreads made from diploid cells should contain 40 chromosomes (1,30). Some of the chromosomal
spreads on a slide, however, will contain fewer than this number (fragmented spreads). This
phenomenon is the result of the physical disruption of the nucleus and the spreading of its
contents such that some of the chromosomes of a given spread are thrown clear of the rest. To
determine whether the number of chromosomes in a particular cell line or transgenic animal is
normal, an average of many spreads showing a strong modal number of 40 must be made.

Mouse chromosomes, unlike their human counterparts, are all acrocentric; i.e., the centromere is
located toward one end of the chromosome. Moreover, the positioning of murine centromeres in
the telomeric region of each chromosome characterizes mouse chromosomes as an extreme
example of this classification, as illustrated in the G-banded metaphase chromosomes presented
in Fig. 2a. This spread is derived from a C57Bl/6J mouse using the protocols described in the text.
The small prominent dark region at the end of each chromosome is the centromeric area. As
shown in Fig. 2, the size distribution of mouse chromosomes is moderate, extending from
elongated bar-like objects to shorter, petite structures.

Abnormalities in a mouse karyotype usually can be recognized at this point in the analysis.
Although changes in the banding pattern of particular chromosomes can occur, they are rare
events and usually are difficult to identify. However, alterations in chromosome number and the
translocation of pieces of one chromosome to another are two of the more common aberrations.
Fluctuation in chromosome number is a constant concern when cells are cultured for extended
periods. Such changes are common and usually produce karyotypes with a chromosome count
greater than 40, including an occasional tetraploid cell (open arrow, Fig. 1). As discussed above,
determination of modal number must be based on an average of many spreads, paying particular
attention to the number and integrity of specific chromosome(s). Translocations of whole
chromosomes or chromosomal fragments are usually recognized by the changes they have
created in chromosome morphology and banding pattern. The suspect chromosome may be
substantially longer or smaller relative to the other chromosomes in the spread. A common type
of abnormality is the fusion of two chromosomes at their centromeres [Robertsonian
translocation (31)]. In addition, translocations involving chromosomal fragments, although less
common, change the length and banding pattern of the chromosomes involved and often occur
with the loss of DNA sequence information, i.e., deleterious events. Many of the karyotypic
abnormalities cited above can be seen in the metaphase spread of Fig. 2b. These chromosomes
are derived from an F9 teratocarcinoma cell line and the arrowheads indicate where Robertsonian
translocations have occurred. The structure enclosed in the square is not a normal chromosome.
Instead, it is a chromosomal fragment containing a centromere, which allows the cell line to stably
maintain this structure. The chromosome marked with an open arrow is an example of
fragmentation that can occur during the spreading process. Here, the centromere of the
chromosome has detached and is held only by a thin strand of chromatin.

Chromosome Identification

The precise identification of mouse chromosomes can be a complicated process even for an
experienced investigator. The task is difficult because of the common morphology shared by each
chromosome (30,32). Characteristics such as relative size and centromere position cannot be used
directly in the identification of most chromosomes. Identification of individual chromosomes rests
primarily on interpretation of the G-banding pattern of that chromosome. This reliance on banding
pattern limits the number of usable metaphase spreads to those with sharp, well-defined bands.
Although chromosomes with suboptimal banding can be identified, the process is difficult and
misassignments can occur.

The cataloging of different chromosomes is based on a standard nomenclature (30) which assigns
a number to each chromosome on the basis of its length and banding pattern. The identification
process is divided into three distinct, consecutive steps:

1. The banding pattern is used to pair the chromosomes and confirm and/or determine the
“gender” of the cells. The net effect of this preliminary characterization is to cut in half the
number of positive identifications that must be made. In addition to the two copies of each
autosome, diploid cells contain either two X chromosomes (female) or one X and one Y
chromosome (male). If female, the chromosomes will generate 20 unique pairs. The X
chromosomes are among the largest and are often heavily stained. If male, only 19 unique pairs of
chromosomes are recovered. The remaining two chromosomes will be dramatically different, as
the Y chromosome is very small and stains homogeneously.

2. The sets of chromosomes are arranged in descending order by size. As a general rule, the size of
an autosome is inversely proportional to the nomenclature number. This preliminary classification
clusters the chromosomes into loosely organized groups. Final chromosome assignments can then
be made among a subset of the total as opposed to all 19 pairs.

3. Individual chromosomes are identified on the basis of their banding pattern which can be
accomplished by comparison with the known karyograms of Fig. 3. The figure contains an idealized
version and five different G-banded examples of each chromosome (mouse strain: C57B1/6J).
Since an average karyotype is made up of chromosomes lacking perfect extension and banding,
the examples illustrate a spectrum of these qualities. Compare the unknown chromosomes to the
examples given in the figure and find a representative karyogram. A systematic comparison of
banding patterns will lead to the proper identification of each chromosome. Note that many of the
mouse strains show C-band (centromere morphology) polymorphisms (e.g., chromosome 8 in the
129 strain compared to C57Bl (1,33,34)), and, as a result, their karyograms may appear somewhat
different.

IN SITU HYBRIDIZATION TO METAPHASE CHROMOSOMES

Currently available technologies allow localization of cloned sequences to specific chromosomes

using nonradioactive DNA probes [e.g., biotin-labeled probes (35)]. The process entails
hybridization of a probe to metaphase chromosome spreads followed by the visualization of
specifically hybridizing sequences. By this means molecular geneticists can determine quickly and
unambiguously the chromosomal location of sequences for which a specific probe exists. The
morphological similarity between mouse chromosomes necessitates banding and identifying the
chromosomes prior to hybridization. Our attempts to reproducibly band and karyotype
chromosomes after hybridization have not been successful; the Giemsa banding pattern is much
more diffuse, making unambiguous chromosome assignment impossible. Unfortunately, the
common experience of many investigators is that giemsa staining slightly lowers the efficiency of
hibridizatión. We feel,however,that when chromosome assignment of the hybridization signal is
required, the small reduction in sensitivity is a necessary evil. The effect, although real, is small
when compared to the influence of target sequence size at the site of hybridization (see below).
Probes labeled with biotin have become the standard of nonradioactive labeling (36-40) with
many manufacturers producing biotin linked to nucleic acid precursors.3 The labeled precursors
are excellent substrates for DNA polymerase I and Klenow fragment and can therefore be used in
both nick-translation and randomprimed reactions. The details of probe synthesis have been
extensively discussed elsewhere (35,41,42). This section, instead, details an in situ hybridization
protocol and discusses the possible probe visualization schemes available. Although many
hybridization protocols exist (37,43-46), they share a number of common features with few
substantial differences.

Materials and Solutions

Immerse coverslips in a 5% (v/v) solution of dimethyldichlorosilane (Sigma) in chloroform;

incubate for 5-10 min at room temperature. Decant this solution and wash the coverslips several
times with 100% ethanol. The solutions are listed roughly in the order of 3 their appearance in the
text. Ultrapure formamide (Boehringer-Mannheim) is deionized with AG501-X8 mixed-bed resin
(Bio-Rad). Stir 5 g of resin with 500 ml of formamide overnight at 4°C. Store aliquots at -20°C.
Denaturation buffer is 70% formamide-1X SSC. In a beaker, rapidly mix 280 ml of deionized
formamide, 100 ml of distilled water, and 20 ml of 20x SSC. Adjust the pH to 7.0 with concentrated
HCl. Store at -20°C. This solution can be reused up to four times before being discarded. Phenol is
equilibrated with 50 mM Tris-HCl, pH 7.4. Carrier DNA is prepared from salmon sperm genomic
DNA (Sigma). Prepare a 10 mg/ml stock in water and acid hydrolyze to small (400-600 bp)
fragments (41). Extract this DNA twice with equal volumes of equilibrated phenol and once with
an equal volume of sevag. [Sevag consists of chloroform and isoamyl alcohol (24:1).] Precipitate
the extracted DNA with 2 vol of ethanol at -20°C. Wash the DNA with 70% ethanol and resuspend
in water at a concentration of 10 mg/ml. Store aliquots at -20°C. Hybridization mix, 1X, is 4X SSC
and 10% dextran sulfate (Pharmacia).

Hybridization and Washing

Preliminary considerations. (a) Slowly warm slides kept at 4°C to room temperature before
unsealing the storage box.

(b) Equilibrate a staining dish containing denaturation buffer in a water bath at 70°C.

(c) Prepare 70,90,95, and 100% ethanol, equilibrating a fraction of each at -20°C.

Hybridization begins with the removal of Giemsa stain through a series of ethanol washes at room
temperature. Destain the slides with three 5min washes in 70% ethanol, followed by a single 5-min
wash in 95% ethanol and a single 5-min wash in 100% ethanol; airdry for 5 min. Denature the
chromosomal DNA by immersing the slides in denaturation buffer at 70°C; incubate for 10 min.
Quench the slides in 70% ethanol cooled to -20°C and incubated at room temperature for 5 min;
follow with two additional 5-min washes in 70% ethanol and two 5-min washes in 95% ethanol. All
washing is performed at room temperature with ethanol at -20°C. Air-dry the slides and use on the
same day.
The appropriate hybridization conditions can vary depending on the nature of the situation. In
most cases, including transgenic animals which have exogenous DNA inserted as a tandem array,
the hybridization buffer is essentially a 1:l dilution of 2X hybridization mix with deionized
formamide and contains 10 pg/ml biotin-labeled probe and 1 mg/ml carrier DNA. Localizing
endogenous sequences with probes derived from large (340 kb) DNA fragments [e.g., a cosmid
clone or a fragment from a YAC (yeast artificial chromosome)] will, however, require modifications
of hybridization conditions. The details of such methods are outlined in Lichter et al. (37) and
include lowering the cation [Na+] concentration to 0.15 M (1X SSC) as well as using 1 mg/ml
mouse genomic DNA as the carrier. The modifications are necessary to suppress cross-
hybridization between repetitive elements found on large probes and related sequences dispersed
throughout the genome.

Depending on the amount of buffer needed (i.e., the number of slides), combine and precipitate
the necessary amounts of probe and carrier DNA with ethanol. The washed and dried pellets
should be taken up in the appropriate amount of deionized formamide and incubated at 70°C for 5
min; quench on ice. At room temperature, add an equal volume of 2~ hybridization mix. Mix
thoroughly and keep at room temperature until use. Hybridization reactions using 30 X 22-mm
coverslips can be conveniently performed with 30 ~1 of hybridization buffer. Use more or less
buffer depending on the size of the coverslip. Apply hybridization buffer directly to airdried slides
warmed to 42°C. The buffer spreads out uniformly when the slide is overlaid with a coverslip.
Place the slides in a culture dish on blot paper saturated with 50% formamide and SSC equivalent
to the concentration in the hybridization buffer. Cover and place the dish in a larger plastic box
that also contains blotting paper saturated with 50% formamide and SSC. Tape this outer box
closed and incubate at 37°C for 316 h. Following hybridization, dip the slides individually into 50%
formamide-2X SSC at 42°C to allow the coverslips to fall off. Immediately place each slide in a rack
under 50% formamide-2X SSC at 42°C. Once all of the slides have been processed, incubate for 5
min. Wash the slides twice more, 5 min each, in 50% formamide-2X SSC at 42°C (i.e., a total of
three washes). The slides should then be washed three more times, 5 min each, at 60°C in 0.1X
SSC. Prepare solutions and other reagents for visualizing the hybridization signal during these
washes.

Visualization of the Hybridization Signal

The enormous advantages offered by biotin-labeled probes are the ease and speed at which the
hybridization signal is visualized. Detection of biotin is accomplished through the high specific
affinity of the small hydrophilic proteins avidin and streptavidin. Attachment of functional motifs
allows the detection of biotin through physical or histochemical means. Therefore, unlike
autoradiographic methods that require dipping in photographic emulsion and weeks of exposure,
visualization of biotin occurs without emulsion and “exposure times” are less than a day.
Functional moieties commonly used for the detection of biotin include horseradish peroxidase
[e.g., (39)] and the fluorochrome fluorescein isothiocyanate [e.g., (37,45)]. Peroxidase
histochemical detection results from the precipitation of diaminobenzidine at the site of
hybridization. This preliminary signal is further enhanced through precipitation of metallic gold
and silver (42). In general, fluorescence-activated detection is more sensitive and easier to
perform. The experience of many groups has shown that the sensitivity of this method is sufficient
to detect target sequences as small as a few kilobases (47-50). If a fluorescence-equipped
microscope is available, fluorescence-activated detection is the method of choice.

Peroxidase histochemical detection [adapted from (39)]. Ligand: horseradish peroxidase (HRP)
conjugated with streptavidin.

MATERIALS AND SOLUTIONS. HRP-conjugated streptavidin can be purchased from ENZO

Diagnostics as a solution in 1X PBS. Subdivide the stock and store at -20°C. Peroxidase detection
buffer can be made by diluting the HRP-conjugated streptavidin stock (1:250) in 4~ SSC-1% bovine
serum albumin (BSA; Sigma); prepare fresh just prior to use. Diaminobenzidine (Sigma) should be
resuspended in water at a concentration of 50 mg/ml. Store as 2-ml aliquots in foil-wrapped tubes
at -20°C. Sodium phosphate buffer is 22 mM sodium dibasic phosphate and 11 mM sodium
monobasic phosphate. Hydrogen peroxide, 30% (v/v), can be purchased from Sigma and stored at
-20°C. A diaminobenzidine enhancement kit is available from Amersham.

Once the hybridization washes are completed, store the slides in 4~ SSC while preparing the
reagents for detection. Slides should be individually removed from the 4~ SSC buffer, briefly
drained, and flooded with peroxidase detection buffer. Line the bottoms of a few petri dishes with
filter paper saturated with 4X SSC and incubate the slides in these closed chambers on a slide
warmer at 37°C for 1 h. Remove unbound ligand with a series of washes. Initially, wash the slides
in 2~ SSC for 5 min, followed by a lo-min wash in 4~ SSC-0.1% Triton X-100. Place the slides into 4~
SSC and keep them there at least 2 min or until needed. In dim light, thaw a 2-ml aliquot of
diaminobenzidine and dilute in 200 ml of sodium phosphate buffer. Prior to use, add 60 ~1 of 30%
hydrogen peroxide, mix, and incubate slides for 3 min. After reaction with diaminobenzidine, wash
the slides three times with distilled water (3 min each). The signal can be amplified using a
diaminobenzidine enhancement kit following the manufacturer’s instructions. Note that the time
of the final silver precipitation reaction is variable and must be determined empirically for each
batch of slides.

Fluorescence-activated detection (37). Ligand: fluorescein isothiocyanate (FITC)-conjugated avidin.

MATERIALS AND SOLUTIONS. FITC-conjugated avidin (Vector Laboratories) is suspended in 1X PBS/

0.02% sodium azide at a concentration of 1 mg/ml and stored at -20°C. Fluorescence detection
buffer is 4~ SSC, 0.1% Tween 20,0.1% BSA, and 5 pg/ml FITC-conjugated avidin. Fluorescence wash
buffer is 4~ SSC0.1% Tween 20. DNA counterstain is 200 rig/ml 4,6diamidino-2-phenylimdole
dihydrochloride (DAPi; Sigma) pepared in 2~ SSC. Labeled slides are protected with a coverslip and
a mounting medium composed of 20 mM Tris-HCl, pH 8.0, 90% glycerol, and 2.3% 1,4-
diazabicyclo[2.2.2]octane (DAPCO anti-fade, Sigma). Once the hybridization washes are
completed, incubate the slides in 4~ SSC-3% BSA at 37°C for 30 min. Thereafter, slides should be
exposed to FITC-conjugated avidin by incubating them in fluorescence detection buffer at 37°C for
30 min. Unbound ligand is subsequently removed by incubating the slides in fluorescence wash
buffer three times at 42°C for 3 min each. Counterstain the slides with DAPi, incubating at room
temperature for 5 min. These preparations should be protected with a coverslip and mounting
medium before examination with a fluorescence microscope

Representative examples of each detection strategy are presented in Fig. 4. Biotin-labeled probes
were prepared and hybridized as described above. The enhanced peroxidase signal shown in Fig.
4a is the consequence of hybridization to exogenous DNA integrated into the genome of a
transgenic mouse line. The fluorescence-activated signal of Fig. 4b results from hybridization to
sequences of a yeast artificial chromosome integrated into the genome of a murine cell line (L
cell). In both examples it is clear that the integration events have occurred at single chromosomal
locations.

COMMENTS The procedures outlined here provide biologists using mice with the necessary
background to assay cells or animals at a cytogenetic level. Such analysis includes the proper
identification of chromosomes (karyotyping) as well as localization of individual sequences to
specific chromosomes. Molecular biologists need to recognize, however, that many of the
procedures presented here are not “recipe-like” protocols. A number of the steps are subjective,
requiring trial and error until appropriate results are obtained. The isolation, and subsequent
cloning, of additional loci should expand the number of workers that will use this technology.
Future insights and modifications to the procedures will surely follow this increased interest in
murine molecular genetics.

FIG. 1. Low-power (10X) inverted microscopic view of freshly spread cell suspension. The slide is
air-dried and unstained. Arrowht indict ate metaphase chromosome spreads that display ideal
spreading and extension. The open arrow indicates a spread derived from a whict L has
spontaneously doubled its chromosome content (i.e., become tetraploid).

FIG. 2. Giemsa-banded metaphase chromosome spreads. (a) Metaphase spread derived from the
bone marrow (methotrexate-synchronized culture) of a female C57B1/6J mouse. Giemsa staining
was performed using G-banding No. 2 as described in the text. (b) Chromosomes prepared from an
F9 teratocarcinoma cell line growing in culture. Giemsa staining was performed using G-banding
No. 1 as described in the text. This cell line contains a number of chromosomal anomalies:
Robertsonian translocations (b), stably transmitted chromosomal remnant (O), chromosome
physically fragmented during the spreading process (-)

FIG. 3. Giemsa (G) band karyograms of mouse strain C57B1/6J. In addition to a standard idiogram,
Giemsa-stained chromosomes exhibit ing a wide range of staining and extension are shown for
each chromosome.

FIG. 4. Visualization of in situ hybridization signal. (a) Detection of the hybridization signal through
peroxidase histochemistry. This chromosome spread is derived from the bone marrow of a
transgenic female mouse containing 20 copies of a 7.7-kb fragment integrated in a tandem array
on chromosome 10. The chromosomes were Giemsa stained and karyotyped prior to the in situ
reaction. The 7.7-kb fragment was biotin-labeled by nick-translation. Hybridization,
posthybridization washes, and visualization of the probe were performed as described in the text.
The arrow indicates the site of hybridization. (b) Visualization of the hybridization signal through
fluorescence-activated detection. The chromosome spread is derived from a murine L-cell line
containing a yeast artificial chromosome (approximately 600 kb) integrated into a single
chromosomal location. A 150-kb region of the YAC was targeted and a biotin-labeled probe was
generated by nick-translation. Hybridization, posthybridization washes, and visualization of the
probe were performed as described in the text. The arrow indicates the fluorescein isothiocyanate
hybridization signal.