BIMM 110    Lecture 16

A. Selection of interspecies somatic cell hybrids

1. The mammalian cell as a microorganism

- cell cultures and cell lines;  mutant phenotypes observable in tissue culture
         auxotrophs, drug-resistance, temperature-sensitive phenotypes,
- mammalian inter- and intra-species cell fusion for complementation testing and mapping
         originally Sendai virus was used as fusogen; nowadays the preference is for polyethylene glycol (PEG)
- selective systems for hybrid cell lines
         HAT medium for selecting against HPRT- and TK- cells; HPRT+ TK+ cells grow normally
         Hypoxanthine, Aminopterin, Thymidine: salvage pathways must be operative for cell to grow in the presence of the folate analogue
- chromosome segregation from interspecies hybrids
         human chromosomes are preferentially lost in hybrids between human and rodent cells
         the number of human chromosomes in such hybrids after a period of transition includes at a minimum the chromosome with the selected marker gene, but in addition a small number of nonselected, randomly retained          chromosomes
- Somatic Cell Hybrid panels

2. Identification of gene products in hybrids

- genetic complementation of known functions
example: HPRT- TK+  human cells fused with HPRT+ TK- mouse cells selected in HAT medium
the resulting hybrid cells will be HPRT+ TK+
- electrophoretic protein variants: e.g. distinction between rodent and human proteins/enzymes on native gels
- immunological techniques

3. Direct mapping of genes using Southern blots with DNA from hybrid panels

  hybrid panels are commercially available

- although a probe will detect homologous sequences from different species, the restriction fragments will generally be different in size

4. Combination of PCR with analysis of hybrid panels

- species-specific oligonucleotide primers (e.g. flanking intron-exon boundaries) can be used to correlate the presence of a specific chromosome with the formation of an expected PCR product, indicating the presence of the template on the chromosome in question
- specificity for the gene of interest comes from the primer annealing to exon sequences
- specificity for the species comes from the primer annealing to intron sequences

5. Subregional mapping

- by the use of chromosome translocations and deletions
- requires very tedious analysis of hybrids with marker chromosomes containing fragments of chromosomes

6. Direct mapping by in situ hybridizations with fluorescent probes (FISH)

- the method of choice today, if a sufficiently long probe is available
- does not require hybrid cells
- requires a probe of  >5 kb (i.e. larger than typical cDNA), but one can get YACs and BACs from commercial source when a probe is identified
- the probe is made with a (nonradioactive) nucleotide analogue, eg. dUTP in which the uracil base is modified by covalent attachment of fluorescent chromophores, or by a small molecule (eg. biotin, hygromycin) that can be detected with specific antibodies or other proteins (eg. avidin) conjugated to fluorescin and other dyes.
- multiple probes (with different chromophores can be used at once to identify chromosomes and even map two syntenic genes with a resolution of a few Mb.

 

more examples will be shown in the lecture (see also RESOURCES FOR MOLECULAR CYTOGENETICS )

 - Davidson, R.L. , F. de la Cruz (eds).(1974). Somatic Cell Hybridization. Raven Press, New York
- Ringertz N.R., R.E. Savage (1976) Cell Hybrids. Academic Press, New York.
- Ruddle, F.H. (1970). Utilization of somatic cells for genetic analysis: Possibilities and problems. In: Symp. of the Int. Soc. for Cell Biol.; vol.9; H.A. Padykula (ed.) Academic Press, New York.