REGULATION OF GENE EXPRESSION IN TRANSGENIC ANIMALS 
Robert E. Hammer, Ph.D., Assistant Investigator 
During the past year, research in Dr. Hammer's 
laboratory has continued to be focused on the reg- 
ulation of eukaryotic gene expression. Specific 
areas of investigation include 1) the function(s) of 
lipoproteins and the low-density lipoprotein (LDL) 
receptor in the development of atherosclerosis, 2) 
the cis-acting DNA elements that direct and modu- 
late cell-specific gene expression, and 3) oncogene- 
induced neoplasia of the cervix and pancreas. 
L Lipoproteins and Lipoprotein Receptors. 
The LDL receptor is a cell surface receptor that 
plays an integral role in the metabolism of choles- 
terol in humans and animals. This receptor binds 
LDL, a plasma cholesterol transport protein, and 
carries it into cells. The LDL receptor is of primary 
importance in cholesterol metabolism, and conse- 
quently the development of atherosclerosis, and is 
a model system for the study of receptor-mediated 
endocytosis. During the past year, Dr. Hammer, in 
collaboration with Drs. Michael Brown and Joseph 
Goldstein (University of Texas Southwestern Medi- 
cal Center at Dallas), has established lines of trans- 
genic mice containing and expressing various 
human LDL receptor minigenes to investigate 1) 
the physiologic consequences of expressing the 
human LDL receptor gene when it is removed from 
its normal feedback control mechanisms, 2) the dif- 
ferences in LDL receptor internalization in various 
somatic tissues and, 3) the nature of receptor-medi- 
ated endocytosis of plasma lipoproteins by perito- 
neal macrophages, a cell type that is a major con- 
stituent of atherosclerotic plaque. 
Mice containing fusion genes consisting of either 
the mouse metallothionein (MT) promoter or 
mouse transferrin promoter/enhancer fused to a 
human LDL receptor minigene were generated, and 
several lines of mice expressing high levels of 
human LDL receptor gene have been established. 
Mice containing either construct express the hu- 
man receptor in a number of tissues, particularly 
the liver, and express the receptor to such levels 
that the animals chronically have little or no LDL in 
their bloodstream. Large numbers of mice exhibit- 
ing this phenotype are being generated and will be 
placed on high-cholesterol diets to examine how 
high internal stores of cholesterol affect endoge- 
nous LDL gene regulation and how such animals 
respond to these high-fat diets. 
In addition to using these mice to investigate the 
effect of chronic overexpression of the LDL recep- 
tor. Dr. Hammer, in collaboration with Dr. Richard 
Anderson (University of Texas Southwestern Medi- 
cal Center at Dallas), is using human LDL receptor 
mice to investigate how the LDL receptor functions 
in various animal tissues. Most tissues of the body 
require cholesterol for such housekeeping func- 
tions as membrane formation. Exactly how the LDL 
receptor contributes to cholesterol transport and 
cellular cholesterol homeostasis in each tissue is 
not known. The low expression of LDL receptors in 
the liver of untreated normal animals has made it 
difficult to study receptor function in situ. Immu- 
nocytochemistry is currently being utilized to map 
the distribution of the human receptor in the liver, 
kidney, and intestine of MT-LDL receptor transgenic 
mice. 
Atherosclerotic plaques are filled with macro- 
phages that have ingested large amounts of choles- 
terol and have been so stuffed with cholesterol es- 
ters that they have become converted into foam 
cells. Plasma lipoproteins are the primary source of 
the cholesterol found in these cells, but the molec- 
ular mechanism of lipoprotein uptake into macro- 
phages is not clear. Recent studies have revealed 
that the LDL receptor is responsible for the uptake 
of p-very low density lipoprotein (P-VLDL) in 
mouse peritoneal macrophages. Because cellular 
uptake of 3-VLDL is mediated through LDL re- 
ceptors, it is important to understand the metabo- 
lism of the LDL receptor in macrophages. A fusion 
gene has been constructed that contains the 
human p^-microglobulin gene enhancer/promoter 
directing the expression of the human LDL re- 
ceptor minigene. Previous experiments in Dr. 
Hammer's laboratory have demonstrated that the 
p2 promoter/enhancer directs expression to many 
cell types, including macrophages. Lines of mice 
containing and expressing this human P^-LDL re- 
ceptor gene have been established, and macro- 
phages will be isolated from these transgenic mice 
and used to investigate lipoprotein uptake and LDL 
receptor metabolism in this important cell type. 
During the past year a second area of investiga- 
tion involving lipoproteins and the development of 
atherosclerosis has been initiated. Lp(a), a variant 
form of LDL, is present in the plasma of humans in 
amounts that vary from undetectable up to 100 
mg/dl. High levels of Lp(a) are strongly correlated 
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