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Giemsa Banding, Mouse Chromosomes
Giemsa Banding, Mouse Chromosomes MT Davisson, The Jackson Laboratory, Bar Harbor, ME, USA
© 2001 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 2, pp 876–877, © 2001, Elsevier Inc.
Identification of individual chromosomes of the laboratory mouse (genus Mus) was virtually impossible until the develop ment of methods for staining metaphase chromosomes to reveal their differential banding patterns. A method for banding mouse chromosomes was first developed using quinacrine mustard fluorescence by Lore Zech and Torbjörn Caspersson in 1969–70. During the early 1970s, several laboratories developed methods using Giemsa stain and various combinations of heat and trypsin treatment, called the ASG (acetic acid–saline– Giemsa) or ASG/trypsin methods. Edward P. Evans was one of the key scientists involved in developing high-quality Giemsa banding (G banding) of mouse chromosomes. The Giemsa stain used in these methods is the same as that traditionally used for staining blood smears. In the mid-1990s, fluorescence banding of chromosomes returned with the use of 4′,6-diamidino-2 phenylindole (DAPI) and related stains to identify mouse chro mosomes with fluorescent in situ hybridization (FISH) gene mapping methods. G banding, however, remains the best method for high-resolution identification of banding patterns in mouse chromosomes and chromosomal aberrations. The basis of all these banding methods appears to be the frequency of A–T versus C–G base pairs in a stretch of chro mosomal DNA. An extensive literature was published during the mid-1970s on ‘chromosomal banding’. It should be noted that even G-banded mouse chromosomes can be difficult for the novice to identify and classify. Although banding patterns of individual chromosomes are nonvariant (except for peri centromeric heterochromatin C bands), they may appear different at different stages of chromosomal contraction. In 1984, Cowell produced a good guide to classification with photographs of mouse chromosomes at different stages of contraction (Cowell, 1984). A standard method for preparing G-banded metaphase chromosomes from living mice is outlined below; details on technique and sources of reagents may be found in Davisson and Akeson (1987). The same method can be used to prepare G-banded chromosomes from any mitotic tissue in the mouse. For example, suspensions of bone marrow cells can be washed out of femurs with a 23–25 gauge needle or solid tissues such as the spleen can be minced and pipetted to obtain cell suspen sions. To prepare metaphase chromosomes from live mice, approximately 70 μl of blood is drawn by retro-orbital or tail vein bleeding and mixed immediately with 0.1 ml sterile sodium heparin (500 USP units per milliliter). Blood is cul tured in 16 mm � 125 mm disposable culture tubes. A 0.2 ml volume of whole blood/heparin mixture is inoculated into 0.95 ml of RPMI 1640 culture medium containing glutamine, Hepes buffer, and gentamicin solution (final concentration, 0.1 mg ml−1), and supplemented with 0.15 ml of fetal bovine serum, 0.1 ml of 750 μg ml−1 lipopolysaccharide (LPS), and 0.1 ml of 60–90 μg ml−1 purified PHA (phytohemagglutinin;
330
concentration determined by a dose–response curve for each batch of PHA). The cultures are incubated at an approximately 45° angle for 43 h at 37 °C in a shaking water bath. Colchicine (0.15 ml of a 50 μg ml−1 solution) is added to each culture for the last 15–20 min. Cells are harvested by centrifugation, resus pension in hypotonic 0.56% (0.75 mol) potassium chloride for 15 min, centrifugation, and fixation in methanol:glacial acetic acid (3:1). After 30 min, cells are centrifuged and resuspended in three sequential washes of the methanol:glacial acetic fixative. The method of slide preparation is important because well-spread metaphases are critical for high-quality G-banded preparations. Precleaned slides are soaked in fixative at least 15 min prior to use. Air-dried metaphases are prepared by dropping a few small drops of cell suspension onto a precleaned slide, allowing it to spread, and then rapidly blowing dry when the drop begins to contract and rainbow colors appear at the edges. Some cytogeneticists believe that spreading is improved by dropping a very small drop of clean fixative onto the preparation just as it starts to dry and allowing the slide to dry in a horizontal position. G bands appear sharper if slides are aged at room temperature for 7–10 days. To prepare G-band chromosomes, slides are incubated in Coplin jars (no more than 5–6 per jar) in 2 � SSC at 60–65 °C for 1.5 h and transferred to 0.9% NaCl at room temperature, and then each slide is rinsed individually in fresh 0.9% NaCl and drained. Thorough rinsing is critical. Slides are stained for 5–7 min in a trypsin–Giemsa solution (1.0 ml Gurr improved Giemsa R66, 45 ml Gurr pH 6.8 phos phate buffer, four drops of 0.0125% trypsin) and transferred to Gurr phosphate buffer diluted 1:1 with distilled water, and then slides are rinsed individually in two changes of buffer– distilled water solution and blown dry. Factors that influence chromosomal response to trypsin treatment and, therefore, G-band quality include chromosome length (contracted chromosomes are more sensitive than elongated ones), chro mosome dryness (recently made preparations are more sensitive than aged ones), and chromosome fixation time (sensitivity is inversely proportional to fixation time or chro mosome hardness).
See also: Chromosome Banding.
References Cowell JK (1984) A photographic representation of the variability in the G-banded structure of the chromosomes in the mouse karyotype. Chromosoma 89: 294–320. Davisson MT and Akeson EC (1987) An improved method for preparing G-banded chromosomes from mouse peripheral blood. Cytogenetics and Cell Genetics 45: 70–74.
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 3
doi:10.1016/B978-0-12-374984-0.00648-3
Giemsa Banding, Mouse Chromosomes
Further Reading Akeson EC and Davisson MT (2000) Analyzing mouse chromosomal rearrangements with G-banded chromosomes. In: Jackson I and Abbott C (eds.) Mouse Genetics and
Transgenics, 2nd edn., pp. 144–153. Oxford: Oxford University Press.
331
Committee on Standardized Genetic Nomenclature for Mice (1972) Standard karyotype of the mouse, Mus musculus. Journal of Hereditary 63: 69–71. Lyon MF, Rastan S, and Brown SDM (eds.) (1996) Genetic Variants and Strains of the Laboratory Mouse, 3rd edn. Oxford: Oxford University Press.
© 2001 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 2, pp 876–877, © 2001, Elsevier Inc.
Identification of individual chromosomes of the laboratory mouse (genus Mus) was virtually impossible until the develop ment of methods for staining metaphase chromosomes to reveal their differential banding patterns. A method for banding mouse chromosomes was first developed using quinacrine mustard fluorescence by Lore Zech and Torbjörn Caspersson in 1969–70. During the early 1970s, several laboratories developed methods using Giemsa stain and various combinations of heat and trypsin treatment, called the ASG (acetic acid–saline– Giemsa) or ASG/trypsin methods. Edward P. Evans was one of the key scientists involved in developing high-quality Giemsa banding (G banding) of mouse chromosomes. The Giemsa stain used in these methods is the same as that traditionally used for staining blood smears. In the mid-1990s, fluorescence banding of chromosomes returned with the use of 4′,6-diamidino-2 phenylindole (DAPI) and related stains to identify mouse chro mosomes with fluorescent in situ hybridization (FISH) gene mapping methods. G banding, however, remains the best method for high-resolution identification of banding patterns in mouse chromosomes and chromosomal aberrations. The basis of all these banding methods appears to be the frequency of A–T versus C–G base pairs in a stretch of chro mosomal DNA. An extensive literature was published during the mid-1970s on ‘chromosomal banding’. It should be noted that even G-banded mouse chromosomes can be difficult for the novice to identify and classify. Although banding patterns of individual chromosomes are nonvariant (except for peri centromeric heterochromatin C bands), they may appear different at different stages of chromosomal contraction. In 1984, Cowell produced a good guide to classification with photographs of mouse chromosomes at different stages of contraction (Cowell, 1984). A standard method for preparing G-banded metaphase chromosomes from living mice is outlined below; details on technique and sources of reagents may be found in Davisson and Akeson (1987). The same method can be used to prepare G-banded chromosomes from any mitotic tissue in the mouse. For example, suspensions of bone marrow cells can be washed out of femurs with a 23–25 gauge needle or solid tissues such as the spleen can be minced and pipetted to obtain cell suspen sions. To prepare metaphase chromosomes from live mice, approximately 70 μl of blood is drawn by retro-orbital or tail vein bleeding and mixed immediately with 0.1 ml sterile sodium heparin (500 USP units per milliliter). Blood is cul tured in 16 mm � 125 mm disposable culture tubes. A 0.2 ml volume of whole blood/heparin mixture is inoculated into 0.95 ml of RPMI 1640 culture medium containing glutamine, Hepes buffer, and gentamicin solution (final concentration, 0.1 mg ml−1), and supplemented with 0.15 ml of fetal bovine serum, 0.1 ml of 750 μg ml−1 lipopolysaccharide (LPS), and 0.1 ml of 60–90 μg ml−1 purified PHA (phytohemagglutinin;
330
concentration determined by a dose–response curve for each batch of PHA). The cultures are incubated at an approximately 45° angle for 43 h at 37 °C in a shaking water bath. Colchicine (0.15 ml of a 50 μg ml−1 solution) is added to each culture for the last 15–20 min. Cells are harvested by centrifugation, resus pension in hypotonic 0.56% (0.75 mol) potassium chloride for 15 min, centrifugation, and fixation in methanol:glacial acetic acid (3:1). After 30 min, cells are centrifuged and resuspended in three sequential washes of the methanol:glacial acetic fixative. The method of slide preparation is important because well-spread metaphases are critical for high-quality G-banded preparations. Precleaned slides are soaked in fixative at least 15 min prior to use. Air-dried metaphases are prepared by dropping a few small drops of cell suspension onto a precleaned slide, allowing it to spread, and then rapidly blowing dry when the drop begins to contract and rainbow colors appear at the edges. Some cytogeneticists believe that spreading is improved by dropping a very small drop of clean fixative onto the preparation just as it starts to dry and allowing the slide to dry in a horizontal position. G bands appear sharper if slides are aged at room temperature for 7–10 days. To prepare G-band chromosomes, slides are incubated in Coplin jars (no more than 5–6 per jar) in 2 � SSC at 60–65 °C for 1.5 h and transferred to 0.9% NaCl at room temperature, and then each slide is rinsed individually in fresh 0.9% NaCl and drained. Thorough rinsing is critical. Slides are stained for 5–7 min in a trypsin–Giemsa solution (1.0 ml Gurr improved Giemsa R66, 45 ml Gurr pH 6.8 phos phate buffer, four drops of 0.0125% trypsin) and transferred to Gurr phosphate buffer diluted 1:1 with distilled water, and then slides are rinsed individually in two changes of buffer– distilled water solution and blown dry. Factors that influence chromosomal response to trypsin treatment and, therefore, G-band quality include chromosome length (contracted chromosomes are more sensitive than elongated ones), chro mosome dryness (recently made preparations are more sensitive than aged ones), and chromosome fixation time (sensitivity is inversely proportional to fixation time or chro mosome hardness).
See also: Chromosome Banding.
References Cowell JK (1984) A photographic representation of the variability in the G-banded structure of the chromosomes in the mouse karyotype. Chromosoma 89: 294–320. Davisson MT and Akeson EC (1987) An improved method for preparing G-banded chromosomes from mouse peripheral blood. Cytogenetics and Cell Genetics 45: 70–74.
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 3
doi:10.1016/B978-0-12-374984-0.00648-3
Giemsa Banding, Mouse Chromosomes
Further Reading Akeson EC and Davisson MT (2000) Analyzing mouse chromosomal rearrangements with G-banded chromosomes. In: Jackson I and Abbott C (eds.) Mouse Genetics and
Transgenics, 2nd edn., pp. 144–153. Oxford: Oxford University Press.
331
Committee on Standardized Genetic Nomenclature for Mice (1972) Standard karyotype of the mouse, Mus musculus. Journal of Hereditary 63: 69–71. Lyon MF, Rastan S, and Brown SDM (eds.) (1996) Genetic Variants and Strains of the Laboratory Mouse, 3rd edn. Oxford: Oxford University Press.