Method: Karyotyping

Rosalie Veile

Principle:

Karyotyping is a valuable research tool used to determine the chromosome complement within cultured cells. It is important to keep in mind that karyotypes evolve with continued culture. Because of this evolution, it is important for the interpretation of biochemical or other data, that the karyotype of a specific sub-line be determined. Numerous different technical procedures have been reported that produce banding patterns on metaphase chromosomes. A band is defined as that part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or lighter. The chromosomes are visualized as consisting of a continuous series of light and dark bands. A G-staining method resulting in G-bands uses a Giemsa dye mixture or Leishman dye mixture as the staining agent. What follows is a brief description of the steps involved in assembling a karyotype.

Time required:

15-30 minutes to cut, arrange, glue and interpret one metaphase spread on a karyotype sheet.

1. Count the number of chromosomes. Solid stained chromosomes or chromosomes treated with a trypsin and giemsa stain can be counted at the microscope with a 100X magnification. However, for analysis such as identification of marker chromosomes or determination of the number of copies of individual chomosomes, it is usually necessary to photograph and print the chromosome spreads. Refer to procedures in this manual for black and white photography and film development. Two prints should be made of each spread. One will be cut for karyotyping; the uncut print serves as a reference if questions arise about the interpretation of a certain chromosome.
2. Cut out each individual chromosome and arrange on a karyotype sheet. Chromosomes are ordered by their length, the position of the centromere, the position of the chromosome bands, and the relative band sizes and distributions. Refer to page 10 of the 1985 ISCN for the human karyotype.
3. In the construction of the karyotype the autosomes are numbered 1 to 22, in descending order of length. The sex chromosomes are referred to as X and Y. The symbols p and q are used to designate, respectively, the short and long arms of each chromosome. There are 7 groups identified in the karyotype and data pertaining to each group can be found on page 7 of the 1985 ISCN.
4. Secure chromosomes in place with glue. Pair the chromosomes closely together and align the centromeres (for easier band comparison and checking for structural chromosome aberrations). If possible, have a second technologist check the interpretation of the karyotype before chromosomes are secured in place.
5. A description of the karyotype should be recorded on the karyotype sheet. First record the number of chromosomes, including the sex chromosomes, followed by a comma (,). The sex chromosome constitution is given next. Any structural rearrangements and additional or missing chromosomes are listed next. Refer to ISCN 1985, table 3 on page 20, for nomenclature symbols and additional information. Other information such as the cell line number, the date karyotype was prepared, the specimen type, and the technologist also should be recorded on the karyotype sheet. Refer to the karyotype sheet

References:

An International System for Human Cytogenetic Nomenclature (1985), S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland).

Worton and Duff (1979), Method Enzymol 58 : 322-344.