Human-Rodent Somatic Cell Hybrids
Recombination Mapping of Meiotic Chromosomes
Fine Structure Mapping of Chromosomes
Human-Rodent Somatic Cell Hybrids - Mapping Human Chromosomes
Somatic cell hybrids are culture lines that contain the entire complement of the mouse genome and a few human chromosomes. These culture lines are developed by mixing human and mouse cells in the presence of the Sendai virus. The virus facilitates the fusing of the two cell types to form a hybrid cell. For a reason that is not entirely known, most, but not all, human chromosomes are lost from the hybrid cell lines. Usually a few human chromosomes are retained. Because the human and mouse chromosomes can be distinguished by chromosome staining techniques, it can be determined which human cells are retained with a specific cell line. Typically, the mouse cell line is mutant for a specific function. Because the hybrids are selected on cells in which the mutant cells could not grow, any surviving hybrid lines would contain the human chromosome that complements (or rescues) the mouse mutation and several other human chromosomes.
Once a complement of hybrid cell lines is developed, a DNA marker can be assigned to specific chromosome. If a DNA marker hybridizes to DNA from a specific line, it must be located on one of the chromosomes in the hybrid. By analyzing the hybridization pattern in a number of lines, the DNA marker can be assigend to a specific chromosome. Let's look at the data in the table below and determine on which chromosomes might DNA markers A, B, and C reside.
| Marker | ||||
|---|---|---|---|---|
| Hybrid Line | Chromosomes | A | B | C |
|
1 |
1 ,4, 5, 8 |
+ | - | + |
|
2 |
1, 3, 7, 8 |
+ | + | - |
|
3 |
2, 4, 6, 7 |
- | + | - |
Becasue the only chromosomes in common between hybrid line 1 and 2 is chromosome one, marker A must be located on chromosome 1. Marker B hybridizes to DNA from lines 2 and 3, and because chromosome seven is the only shared chromsome between the two lines, the marker must be on that chromosome. Finally, the fact that the only unique chromosome in hybrid 1 is number 5, marker C must be located on that chromosome.
These lines are not only useful for DNA markers, they can be used for any biochemical phenotype that can be screened at the cellular level. This includes enzymes as well as human cell surface proteins that can be screened in some biochemical manner.
Two extensions of the technique allow for finer analysis of the linkage between genes. Individual human chromosomes can be selected by a technique called flourescence-activated chromosome sorting. The single chromosome can then be added to a mouse cell line, and either the whole chromosome is taken up intact or fragments of the chromosome become integrated into the mouse chromosome. Alternatively, a human cell line can be irradiated with X-rays before fusion. Most often the fragments become incorporated into the mouse chromosome, although a fragment may be maintained as its own partial chromosome. These lines are called radiation hybrids.
As with the cell lines described above which contained a series of chromosomes, these cell lines can be used to determine not only chromosomal locations, but linkages as well. If two markers are continually found on the same fragment of chromosome, then it can be determined that the markers themselves are linked. To determine order, though, a large number of different cell lines must be developed. Each of the cell lines would contain either a different complement of human chromosomes, or different fragments of the same chromosome. By analyzing a large number of these lines, marker orders can be determined for all the chromosomes. For radiation hybrids, though, DNA markers are almost exclusively used for developing maps.
Copyright © 1997. Phillip McClean