Transgenic Mouse Model Of Mutation Overall Level Of Effect Of Apoe Gene Expression

Apolipoprotein E (apoE) plays a key role in lipid metabolism, especially in cholesterol transport, uptake and redistribution. Within nervous system, apoE is involved in the growth and development, signal transduction, also in maintaining synaptic integrity during injury or aging. Additionally apoE is found to be correlated to the modulation of carcinogenesis and immunodeficiency of the body. The purpose of this project is to establish two human apoE variants transgenic mouse models (mutant apoE4 and apoE7) at first. Then using the mouse line investigated the influences of mutant apoE in lipid metabolism. In addition, the abnormalities of learning and memory ablilities, the cancersusceptibilities and some other characteristics of these models were also observed.The eukaryotic expressing plasmids pME4 and pME7 were constructed by cloning human apoE genomic DNA fragment into pBx322/MT to make the genomic DNA under the control of mouse MT-I promoter. The transient and stable expression of human apoE variants were examined in NIH/3T3 cells following Lipofectamine mediated gene transfer. The results showed that NIH3T3 cells were capable of correctly transcribing, translating and expressing biological human apoE proteins. The levels of human apoE which expressed stably in NIH3T3 cells was found to correlate with the copy numbers of apoE gene at the mouse chromosome. Some metals such as Zn⁺⁺ could enhance the expression level of apoE by 45-60%. These results suggested that the expression of human apoE gene was not restricted to human cells, whereas it can be also expressed in mouse cells and regulated by MT-I promoter correctly. Therefore, these approaches at cellular level provided the base for establishing apoE transgenic mouse models.Two human apoE variants (apoE4 and apoE7) transgenic mouse models were constructed with microinjection technique. Among 125 mice, three apoE7 founders tg1, tg2 and tg3, two apoE4 founders tg4 and tg5 were screened by Southern blot analysis. Furthermore, F1-F5 generation of transgenics were obtained through mating male and female among transgenics or between transgenics and normal mice.