Abstract
Adenosine deaminase (ADA) has been shown by genetic and biochemical evidence to be essential for the development of the immune system. In order to study ADA gene structure, regulation and expression, we have isolated mouse cell lines which contain amplified copies of ADA genes. In some cell lines ADA accounts for more than 75% of the soluble protein, representing an increase of 11,400 fold over parental cells. A cDNA library was constructed in pBR322 using an amplified cell line. Functional mouse ADA cDNA clones were isolated by genetic complementation of ADA-deficient E. coli. Analysis of plasmids containing functional ADA cDNA sequences suggested that ADA expression resulted mainly from β-lactamase/ADA fusion proteins. The nucleotide sequence of a 1.65 kb insert was determined and found to contain a 1.056 kb open-reading frame (ORF). When this ORF was inserted into a bacterial expression vector, only a single band of murine ADA was detected upon starch gel analysis. This ORF was placed in a mammalian expression vector and introduced into COS cells where a high level of authentic murine ADA was obtained. A variety of rodent cell lines have been transformed using this vector. Helper-free preparations of retroviruses have been prepared which are capable of transducing functional ADA cDNA into cultured mammalian cells. These retroviral vectors should allow us to introduce the gene into hematopoietic stem cells.
Article PDF
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ingolia, D., Yeung, CY., Shoemaker, C. et al. EXPRESSION OF MURINE ADA CDNA IN E. COLI AND MAMMALIAN CELLS: 90. Pediatr Res 19, 758 (1985). https://doi.org/10.1203/00006450-198507000-00110
Issue Date:
DOI: https://doi.org/10.1203/00006450-198507000-00110