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T Y Chang

Publications and source records attributed to T Y Chang.

9 recordsLinked to original sources
PubMed1991

High-efficiency stable gene transfection using chloroquine-treated Chinese hamster ovary cells.

We describe a highly efficient stable gene transfection procedure for Chinese hamster ovary (CHO) cells using a modification of the calcium phosphate-DNA coprecipitation method. We have found that treatment of CHO cells with chloroquine increases the efficiency of gene transfer by up to 20-fold (from approx. 0.01% to approx. 0.2%) when transfection is done using the pSV2-neo plasmid. The optimized transfection procedure requires that CHO cells to be incubated with calcium phosphate-DNA coprecipitate and chloroquine (100 microM) for a total of 16 h. By using high-molecular-weight human genomic DNA as a DNA source for transfection, we show that this procedure is equally efficient for stably transferring a much larger gene, such as the 49-kb human hypoxanthine phosphoribosyltransferase gene.

Animals↗
PubMed1978

Mammalian cell mutant requiring cholesterol and unsaturated fatty acid for growth.

A mutant requiring both cholesterol and oleate for growth has been isolated from mutagenized Chinese hamster ovary cells. By comparison with wild-type cells, sterol and unsaturated fatty acid biosynthetic activities in the mutant cells grown in fetal calf serum medium appear to be nearly intact. However, whole-cell radioactive acetate, mevalonate, dihydrolanosterol, and stearate incorporation studies show that sterol synthesis from acetate, lanosterol demethylation, and fatty acid desaturation are defective in the mutant cells grown in delipidated serum medium. In vitro enzyme assays with crude cell extracts demonstrated that beta-hydroxy-beta-methylglutaryl-coenzyme A reductase is not induced in the mutant. These experiments were substantiated by gas/liquid chromatographic analyses which showed the sterol content and the percentage unsaturated fatty acids in mutant cells to be drastically reduced when the cells are grown in delipidated serum medium. A spontaneous revertant exhibiting prototrophic growth in lipid-free medium has been isolated from 50 X 10(6) mutant cells. All three defects in this revertant reverted back in parallel, which suggests that these three biosynthetic activities may be controlled by a common regulatory mechanism.

Cell Division↗
PubMed1977

Isolation and partial characterization of a cholesterol-requiring mutant of Chinese hamster ovary cells.

A sterol-requiring mutant has been isolated from mutagenized Chinese hamster ovary cells. This mutant grows normally only when cholesterol is present in the medium. Cell lysis occurs within 3 days in the absence of cholesterol. The frequency of reversion of this mutant to prototrophic growth is low (less than or equal to 10(-6). Whole cell pulse experiments with [14C]acetate or [3H]mevalonate indicate that the rate of synthesis of digitonin-precipitable material is greatly diminished in the mutant cells as compared to that in normal Chinese hamster ovary cells. Enzyme assays in vitro with crude cell extracts show that the biosynthetic conversion of mevalonate to squalene and the conversion of squalene to lanosterol are not impaired in the mutant cells. Gas-liquid chromatographic analyses of radioactive sterol composition after whole cell pulse experiments with [3H]squalene and with [3H]anosterol suggest that the fundamental enzymatic defect of the mutant is at the stage of lanosterol demethylation. When cells were grown in serum-free medium, lanosterol and dihydrolanosterol accumulated intracellularly in the mutant cells before cell lysis occurred; neither of these two intermediary sterols was detected in the wild-type cells grown under the same condition.

Animals↗
PubMed1976

Isolation and characterization of an unsaturated fatty acid-requiring mutant of cultured mammalian cells.

An unsaturated fatty acid-requiring mutant derived from Chinese hamster ovary (CHO) cells has been isolated and characterized. This mutant grows normally when oleate or other unsaturated fatty acids are supplemented in the growth medium. Unlike the wild-type CHO cells, growth stops when medium is deprived of unsaturated fatty acid. Whole cell pulse experiments with [14C]acetate or [14C]stearate indicate that the mutant is defective in unsaturated fatty acid synthesis. Enzyme assays in vitro show that the enzymatic defect of the mutant is localized to the microsomal stearoyl-CoA desaturase.

Acetates↗
PubMed1975

Mechanized enzymatic determination of triglycerides in serum.

A procedure for enzymatic determination of serum triglycerides [Clin. Chem. 19, 476 (1973)] has been adapted for use in continuous-flow analysis (Technicon AutoAnalyzer). A very simple manifold is used; serum is incubated at 37 degrees C with the lipase and alpha-chymotrypsin in potassium phosphate buffer (0.1 mol/liter, pH 7, containing 1.50 g of bovine serum albumin per liter). The liberated glycerol is dialyzed against the complete glycerol reagent. The change in absorbance at 340 nm resulting from oxidation of NADH is proportional to the dialyzed glycerol. The same manifold can be used to determine preformed glycerol if the hydrolyzing enzymes are omitted. The hydrolysis is complete, as shown by the use of equivalent glycerol standards. No prior treatment of the samples is necessary. Assays are run at 60 per hour in the AutoAnalyzer l, 80 per hour in the AutoAnalyzer ll. Results with both instruments for 150 samples correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure.

Autoanalysis↗