In this article we will discuss about the mechanism of formation of somatic cell hybrids.
It had been known since the 60’s that somatic cells from the same or different species in culture could spontaneously fuse to form polyploid cells. The product of fusion was called homokaryon if the two parental cells came from the same species, and heterokaryon or somatic cell hybrid if the fusion was interspecific. The hybrid cells could divide by mitosis and proliferate and thus could be maintained in culture (Fig. 21.7).
Two further technical advances made human gene mapping by somatic cell hybrids possible. In 1962 Okada discovered that inactivated Sendai virus could greatly increase the rate of cell fusions. Since then several agents causing cell fusion have been tried among which polyethylene glycol has some advantages. The exact mechanism of cell fusion is not known.
In the case of UV-inactivated Sendai virus, it seems that the virus absorbs to the cell surface leading to agglutination of cells. The protein coat of the virus forms the connecting bridge between the cells.
The membranes of the two cells swell into this region and when they come in contact are dissolved. The cell contents mix up, the nuclei fuse and a heterokaryon is formed. When cell fusion is mediated by polyethylene glycol, the two cell membranes directly come in contact.
The second technical advancement was the finding that when sub-lines of hybrid cells are maintained in culture, there is gradual and preferential loss or retention of specific chromosomes. The association between the retention of a genetic marker and that of a specific human chromosome could be determined.
In mouse-man hybrids, most of the chromosomes of the mouse are retained. By using a selective medium which allows growth of cells having a particular chromosome, it is possible to locate genes on a specific chromosome. This technique has been extensively applied for human gene mapping.
The following example illustrates the technique used by Littlefield (1964) for assigning genes to specific chromosomes. He used deletions and mutations affecting enzymes involved in the purine and pyrimidine salvage pathways, namely thymidine kinase (TK) and hypoxanthine guanine phosphoribosyl transferase (HGPRT). There are two pathways for DNA synthesis.
In the first, which proceeds under normal conditions, DNA is synthesised from simple organic molecules and the necessary enzymes. The second is the alternate or salvage pathway which utilizes nucleotide precursors for DNA synthesis.
The salvage pathway is followed only if the first pathway is blocked by an antimetabolite (for example aminopterin) or by a mutation. Two enzymes are necessary for the salvage pathway, HGPRT and TK. If even one of the two enzymes is absent, DNA synthesis cannot take place by the salvage pathway.
Cells from a mutant mouse cell line deficient in the enzyme TK (i.e. TK–/HGPRT+) were mixed with cells from a human line deficient in HGPRT (i.e., TK+/HGPRT–) and allowed to grow on minimal medium. Under appropriate conditions the cells fused to form hybrid cells KT+/TK–; HGPRT+/HGPRT–). It should be noted that in the hybrid cells there is one normal allele for the enzyme TK (from the human cell line) and one normal allele for HGPRT (from the mouse cell line).
All the cell lines could grow on a minimal medium. The mouse cell line (TK–) is not able to grow on a medium containing thymidine as the cells are deficient in TK. Similarly the human cell line (HGPRT–) cannot grow on a medium containing hypoxanthine due to lack of the enzyme HGPRT.
It is also noteworthy that neither of the mutant cell lines is able to grow if aminopterin, an antimetabolite is present in the medium. Aminopterin acts by inhibiting the enzyme folic acid reductase which catalyses the synthesis of reduced folate. The latter is required in the various steps of the normal pathway leading to the synthesis of DNA.
In the presence of aminopterin therefore, DNA is synthesised through the salvage pathway, but only if the enzymes TK and HGPRT are available. The mouse-man hybrid cells are thus able to grow in presence of aminopterin if thymidine and hypoxanthine are present in the medium.
There is a selective medium which allows growth of the hybrid cells but inhibits the parental cells. This medium contains hypoxanthine, aminopterin and thymidine and is called HAT medium. The hybrid cells will proliferate on HAT medium to form colonies as they alone have genes for both TK and HGPRT. Colonies of hybrid cells can be sub-cultured and cloned for mapping genes.
Sub-lines which show progressive loss of human chromosomes are maintained. Only those cells that retain the specific chromosome having the gene for thymidine kinase would survive in HAT medium. It was found out that only cells retaining chromosome 17 could grow on HAT medium. Obviously the gene for thymidine kinase is located on chromosome 17.
The mouse-man cell hybrids have the following advantages for studies on gene mapping:
1. Preferential loss of human chromosomes.
2. Availability of cell lines with identifiable human phenotypes different from those in rodents.
3. The apparent distinction between rodent and human chromosomes in interspecific hybrids.
4. That both rodent and human genes are simultaneously expressed in the cell hybrids and the product proteins of each can be identified individually.
5. The linkage groups on mouse chromosomes are known.