Chromosome Preparation From Cultured Cells

Summary

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Abstract

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births^1,2, 60-80% of all miscarriages^3,4, 10% of stillbirths^2,5, 13% of individuals with congenital heart disease⁶, 3-6% of infertility cases², and in many patients with developmental delay and birth defects⁷. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance^8,9.  Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents^10-13.

Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)^14,15.

Introduction

Chromosome analysis is a conventional technique utilized worldwide to diagnose chromosome instability and rearrangements leading to genetic disorders and malignancy^1,2,8,9. In addition, a higher resolution for the diagnosis and research of constitutional and cancer-acquired genetic abnormalities can be achieved with the combination of the classical cytogenetic procedures and molecular cytogenetic methodologies such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)^14,15. More recently, these techniques have been utilized for the evaluation of chromosome instability associ....

Protocol

1. Chromosome Harvesting of Adherent Cells

1. Standard Protocol
   1. Grow cells according to specific cell culturing conditions. When the cells have reached logarithmic phase (80% confluency), add 10 μl/ml of Colcemid to the cell culture flask. A minimum of 2 x 10⁶ cells is recommended.
   2. Incubate cells at 37 °C in a 5% CO₂ incubator for 45 min. Using a sterile pipette, transfer media from cells into a 15 ml conical tube. Set aside.
   3. Gently wash the cells by adding 2 ml of HBSS Buffer into the flask. Swirl buffer and then remove using a pipette. Discard.
   4. Add 1 ml of tryps....

Results

High quality metaphase spreads are essential for chromosome analysis. A successful assay yields chromosomes which are well spread and of suitable chromosome morphology. Properly G-banded chromosomes contain the characteristic light and dark banding patterns.

Discussion

We have utilized the present procedure for chromosome isolation from cells of various organisms, including various cell types obtained from human, Rhesus macaques, rats, and mice^11,20-22. The standard protocol is provided, but certain key steps and variables may need to be adjusted for these diverse types of cells. Several specific steps are crucial in both the chromosomal preparation and G-banding to ensure the best possible quality of the results. One of the variables which can affect the assay is the Colcem.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KaryoMAX Colcemid	Gibco	15212-012
HBSS Buffer	Gibco	24020-117
0.5% Trypsin EDTA 10x	Gibco	15400-054
Permount	Fisher Scientific	SP15-100
Buffer Tablets "GURR"	Gibco	10580-013
Geimsa Stain	Ricca Chemical Company	3250-16
Centrifuge 5810	Eppendorf
Methanol	Caledon Laboratory Chemicals	6700130
Glacial Acetic Acid	Krackeler Scientific Inc.	11-9508-05