Markers for Gene Expression in Cultured Cells from the Nervous System

Samuel H Wilson,Bruce K Schrier,John L Farber,Edward J Thompson,Roger N Rosenberg,Arthur J Blume,Marshall W Nirenberg

doi:10.1016/s0021-9258(19)45227-7

Abstract

Abstract Methods are presented for preparation of extracts from cultured cells from the nervous system and for study of choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, and catechol O-methyltransferase activities. These enzyme activities are markers that can be used for studying gene expression in neurons. The methods are sufficiently sensitive so that all assays can be performed with protein harvested from one Petri dish. Activities of the marker enzymes were assessed in surface cultures of newborn mouse brain cells, and in glial and nonbrain cell lines. Low activities of choline acetyltransferase, acetylcholinesterase, and glutamate decarboxylase were detected in all the cells tested. All of these activities, and particularly glutamate decarboxylase, were higher in cultured brain cells from newborn animals than in non-neuronal cell lines. Glutamate decarboxylase activity in glial cells and in brain cells was inhibited more than 95% by 1 mm amino-oxyacetic acid.

Highlights

These enzyme activities are markers that can be used for studying gene expression in neurons
In evaluation of the assays neuroblastoma clone N-18 homogenate was used for acetylcholinesterase and catecho 0-methyltransferase assays; mouse brain extracts were used for evaluation of the assays for choline acetyltransferase and glutamate decarboxylase, these assays were applicable to cell culture extracts
The range of linearity with protein for neuroblastoma choline acetyltransferase was about half that obtained with mouse brain homogenates

Summary

Methods

Isolation and Culture of Newborn Mouse BrairbCellsWhole brains from newborn Balb/c (National Inst,it,utes of Health stock) mice were placed in Solution D (137 IIW NaCl, 5.4 mm KCl, 0.17 mM Na2HP04, 0.22 mM KH,PO+ 5.5 ml~ glucose, and 5.9 mM sucrose) pH 7.2, 340 mosM (modified Puck’s Dl solution (12)) at O-4”, weighed, washed several times with Solution D, and minced to about l-mm[8] pieces with iris scissors. The minced tissue was subjected to two 15.min treatments with 0.25% crude trypsin (Nutritional Biochemicals 1:250 or Difco 1:300) in Solution D (100 ml per g of tissue) at. Tissue pieces were allowed to sediment and the dissociated cells t,hat remained suspended were collected by decantation. Tryptic activity in both the cell suspension and undissociated tissue was inhibited by the addition of an equal volume of growth medium containing 10% (v/v) fetal bovine serum (Colorado Serum Co.). Usual cell recoveries for dissociation of newborn brain were 90 to 100 X lo[6] cells per g of tissue (98 to 100% viable). Cells were inoculated at 10’ viable cells per 150.mm Falcon polystyrene Petri dish (145 cm[2] surface area) in 20 ml of D~\IEM

Results

Discussion

Conclusion