Abstract
Previous studies have demonstrated the usefulness of flow cytometry in the analysis of dihydrofolate reductase (EC 1.5.1.3) gene amplification. However, this powerful and potentially sensitive method for analyzing gene expression in individual cells has not seen widespread use. This is due in part to the difficulty of producing fluorescent methotrexate (Fluo-MTX), which is needed to label dihydrofolate reductase in vivo, in yields higher than 1% and of sufficient purity to give low nonspecific backgrounds by the published procedures. We have significantly improved the synthesis of Fluo-MTX to obtain rapidly a chromatographically pure product in 20% yields. In addition, we have found that cell volume is a variable which makes direct comparisons of fluorescence intensity between cell lines difficult. In order to circumvent this problem, we have improved flow cytometric analysis to measure the fluorescence specific intensity of individual cells. A survey of various cells commonly used for gene transfer shows a significant variability in the efficiency with which they are labeled with Fluo-MTX, which appears to be due to variations in their ability to transport this reagent.
Highlights
Background fluorescencewas estimated presence of20 p~ MTX at the same time as rials and Methods”).after labeling cells in Fluo-MTX
SV40 sequences derived from pSV2-DHFR (Lee et al, 1981);El,mouse mammary tumor virus long terminal repeats sequences from pMDSG (Lee et al, 1981); 8, PyrB gene of Escherichia coli coding for the bacterial aspartate transcarbamylase'; B,BstEII/EcoRI restriction fragment from cosmid pcos2EMBL (Poustka et al, 1984)carrying the Cos site of phage x; B, BclI/HphI fragment of polyoma virus carrying the viral origin of DNA replication and the early transcription promoter/enhancer
Since the dihydrofolate reductase gene present in the plasmid which was used to obtain pCDP-3 transformants was derived from pSV2-DHFR and the latter encodes a kinetically normal enzyme, we infer that the mutation leading to the observed change in MTX binding characteristics must have occurred during cloning
Summary
SV40 sequences derived from pSV2-DHFR (Lee et al, 1981);El,mouse mammary tumor virus long terminal repeats sequences from pMDSG (Lee et al, 1981); 8, PyrB gene of Escherichia coli coding for the bacterial aspartate transcarbamylase'; B,BstEII/EcoRI restriction fragment from cosmid pcos2EMBL (Poustka et al, 1984)carrying the Cos site of phage x; B, BclI/HphI fragment of polyoma virus carrying the viral origin of DNA replication and the early transcription promoter/enhancer. 1980) expresses the donated genes at the expected levels (10-50% of the wild type dihydrofolate reductase activity) when transformed by different dihydrofolate reductase minigenes, but does not generate wild type transformants which can be labeled with Fluo-MTX at a significant level. Dihydrofolate reductase-deficient DG44 cells are well suited for analyzing expression and amplification of donated dihydrofolate reductase genes using Fluo-MTX and flow cytometry. We have analyzed a variety of cell lines commonly used for gene transfer by flow cytometry after labeling with Fluo-MTX andhave found that they differ significantly in their ability to be labeled with Fluo-MTX. The implications of this result are discussed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
