Roots. Use of the HPRT gene and the HAT selection technique in DNA-mediated transformation of mammalian cells: First steps toward developing hybridoma techniques and gene therapy
✍ Scribed by Waclaw Szybalski
- Publisher
- John Wiley and Sons
- Year
- 1992
- Tongue
- English
- Weight
- 803 KB
- Volume
- 14
- Category
- Article
- ISSN
- 0265-9247
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
In 1956, I decided to apply my experience in microbial genetics to developing analogous systems for human cell lines, including the selection of mutants with either a loss or gain of a biochemical function. For instance, mutants resistant to azahypoxanthine showed a loss of the HPRT enzyme (hypoxanthine phosphoribosyl transferase), whereas gain of the same enzyme was accomplished by blocking de novo purine biosynthesis with aminopterin, while supplying hypoxanthine and thymine (HAT selection). Using HAT selection, we: (i) genetically transformed HPRT^−^ mutant cells to HPRT^+^ wild type by using DNA extracted from HPRT^+^ cells, and (ii) selected HPRT^+^ hybrid cells by fusing HPRT^−^ D98/AH2 cells with skin cells. These approaches, which we dubbed in 1962 as a [first step toward gene therapy], contributed to the later development of (i) cell fusion techniques, (ii) the development of monoclonal antibodies, (iii) routine transformation of mammalian cells with cloned genes, and (iv) methods for creating transgenic organisms.