SOMATIC CELL HYBRIDISATION SOMATIC CELL HYBRIDISATION

INTRODUCTION It involves the fusion of somatic cells growing in vitro. First demonstrated with mouse cells by G.Barski (1960). Somatic cell hybrids can be produced between two cell of same species or two cells from different species. For ex. Mouse-human hybrids. Used for mapping human gene.

. Human – mouse hybrids are usually made using established mouse cell culture lines and human fibrocytes or leukocytes. Important features of human-mouse hybrids:- 1. Mouse-human chromosomes are easily distinguished. 2.Both sets of chromosomes of human and mouse are expressed in hybrid cells.

. involves fusion of somatic cells growing in vitro. Genes are shown to be located on specific chromosomes by correlating the phenotypes ex. Enzyme activities controlled by these genes with presence of individual human chromosomes in hybrid cell clones. For example, human cell make enzyme thymidine kinase (TK+) were hybridized with mouse cell make enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT).

Cell fusion produces binucleate hybrid cells or heterokaryons. Frequency of spontaneous fusion is very low - increased by addition of ultraviolet-inactivated sendai virus or chemical polyethylene glycol- agent stimulate cell fusion by increasing cell contact and altering cell membrane. Cell fusion produces binucleate hybrid cells or heterokaryons. Cell fusion is followed by nuclear fusion to produce uninucleate hybrid cell or synkaryons hybrid cell clones will grow on HAT medium. Each of 24 different chromosomes can identified using G-banding. .

BIOCHEMICAL BASIS OF SELECTION PROCEDEURE Involves two important pathways for biosynthesis of nucleotides require for DNA synthesis i.e. de novo pathway and salvage pathway. de novo pathway : major pathway by which nucleotides are synthesized from simple sugars and amino acids or this pathway is blocked by antimetabolite aminopterin. If de novo pathway is blocked, then second pathway called salvage pathway comes.

Salvage pathway: HGPRT catalyzes the conversion of hypoxanthine to guanine and thymidine kinase catalyzes the phosphorlyation of thymidine. Enzyme TK+ and HGPRT+ are necessary for salvage pathway.

RECOMBINAtion FREQUENCY . RECOMBINAtion FREQUENCY Recombination fraction is measure of distance between the two loci. Two genes that undergo independent assortment have recombination frequency of 50 percent and are located on non homologous chromosome or far apart on same chromosome – unlinked. Genes with recombination frequencies less than 50 percent are on same chromosome –linked.

. TO CALCULATE RECOMBINANT FREQUENCY: The Recombination frequency for interval from a gene to centromere is one-half the frequency of asci that exhibit second-division segregation patterns for the alleles of the gene.

For example: