Patyal Pankaj, Fil Daniel, Hamdan Hamdan, Wight Patricia A
Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
Front Cell Neurosci. 2023 Jan 11;16:1087145. doi: 10.3389/fncel.2022.1087145. eCollection 2022.
Much of what is known about the mechanisms that control the developmental expression of the myelin proteolipid protein gene () has been attained through use of transgenic animal models. In this study, we analyzed expression of related transgenes which utilize genomic DNA from either human or mouse to drive expression of a reporter. Human () sequence span either the proximal 6.2 or 2.7 kb of 5'-flanking DNA to an internal site in Exon 2, while those from mouse comprise the proximal 2.3 kb of 5'-flanking DNA to an analogous site in Exon 2. Transgenes with sequence were named, in part, to the amount of upstream sequence they have [6.2hPLP(+)Z/FL and 2.7hPLP(+)Z]. The transgene containing mouse sequence is referred to here as mPLP(+)Z, to denote the species origin of DNA. Mice which harbor the 6.2hPLP(+)Z/FL transgene were used as a model system to investigate the developmental expression of splice variants that incorporate supplementary exons from what is classically defined as intron 1. While expression of the splice variants were detected in brain through RT-PCR analysis, they are present at much lower levels relative to the archetypal (classic) transcript. Additionally, we show that mice which harbor the 6.2hPLP(+)Z/FL transgene demonstrate wide-ranging expression throughout brain at P2, whereas expression of mPLP(+)Z is quite limited at this age. Therefore, we generated new transgenic mouse lines with the 2.7hPLP(+)Z transgene, which contains sequence orthologous to just that in mPLP(+)Z. Of the seven lines analyzed, six showed higher levels of 2.7hPLP(+)Z expression in brain at P21 compared to P2; the other line expressed the transgene, only weakly, at either age. This trend, coupled with the robust expression observed for 6.2hPLP(+)Z/FL at P2, suggests that the distal 3.5 kb of 5'-flanking DNA specific to 6.2hPLP(+)Z/FL contains regulatory element(s) important for promoting early postnatal expression in brain.
关于控制髓磷脂蛋白脂蛋白基因（）发育表达机制的许多知识都是通过使用转基因动物模型获得的。在本研究中，我们分析了相关转基因的表达，这些转基因利用人类或小鼠的基因组DNA来驱动报告基因的表达。人类（）序列跨越5'-侧翼DNA的近端6.2或2.7 kb至外显子2中的一个内部位点，而来自小鼠的序列包括5'-侧翼DNA的近端2.3 kb至外显子2中的一个类似位点。含有序列的转基因部分根据它们所具有的上游序列量命名[6.2hPLP(+)Z/FL和2.7hPLP(+)Z]。含有小鼠序列的转基因在这里称为mPLP(+)Z，以表示DNA的物种来源。携带6.2hPLP(+)Z/FL转基因的小鼠被用作模型系统，以研究包含来自经典定义为内含子1的补充外显子的剪接变体的发育表达。虽然通过RT-PCR分析在脑中检测到了剪接变体的表达，但它们相对于原型（经典）转录本的水平要低得多。此外，我们表明，携带6.2hPLP(+)Z/FL转基因的小鼠在出生后第2天在整个脑中表现出广泛的表达，而mPLP(+)Z在这个年龄的表达相当有限。因此，我们用2.7hPLP(+)Z转基因产生了新的转基因小鼠品系，该转基因包含与mPLP(+)Z中序列直系同源的序列。在分析的七个品系中，六个在出生后第21天的脑中2.7hPLP(+)Z表达水平高于出生后第2天；另一个品系在两个年龄时转基因表达都很弱。这种趋势，再加上在出生后第2天观察到的6.2hPLP(+)Z/FL的强劲表达，表明6.2hPLP(+)Z/FL特有的5'-侧翼DNA的远端3.5 kb包含对促进出生后早期脑中表达重要的调控元件。
