Section 6
Chapter 5,591

Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells

Deisseroth, A.; Hendrick, D.

Cell 15(1): 55-63


ISSN/ISBN: 0092-8674
PMID: 279411
Accession: 005590337

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The genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, was used to establish a method for the transfer of human .alpha.-globin genes into mouse erythroleukemia cells. Mouse [MEL] erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the 2 hybrid cell populations and assayed by molecular hybridization for sequences coding for human .alpha.-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human .alpha.-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human .alpha.-globin mRNA and human .alpha.-globin chains. Hybrid cells counter-selected in DAP and lacking human chromosome 16 were devoid of detectable levels of human APRT, .alpha.-globin mRNA and .alpha.-globin chains. Transfer of human chromosome 16 into the MEL cell is possible using a chromosome-dependent, APRT-mediated method of gene transfer. Using this system in which expression of the human .alpha.-globin gene occurs, an earlier assignment of the human .alpha.-globin gene to human chromosome 16 was also confirmed. This system may be of further use in identifying genetic elements governing expression of the human .alpha.-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.

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