SICKLE CELL ANEMIA MOUSE MODEL

这是制造镰刀状贫血小鼠模型的继续努力。在过去的几年中，我们成功地构建了一种镰状细胞小鼠，其中成年小鼠产生的主要血红蛋白是HBS。该模型与已有的β/S珠蛋白转基因模型不同的是，该模型位于一个YAC内，该YAC包含基因座控制区和整个β-珠蛋白基因簇。由于珠蛋白基因的控制区以及假定的增强子和消音器序列处于天然环境中，这些小鼠可能会更接近人类，以测试影响HBF产生的药物。这些小鼠制备过程中的一个问题是，产量极低。这可能是因为这些小鼠在胚胎早期关闭了伽马珠蛋白基因的表达，而高水平的HBS在子宫中是不能容忍的。我们在未来几年的目标是使用几种方法让大脑镰状细胞小鼠存活到新生儿期。首先，我们将继续培育它们，看看是否能获得更多存活的镰状细胞小鼠。其他人的经验是，尽管由于围产期死亡而在获得镰状细胞小鼠方面最初存在差异，但经过反复繁殖，最终可能由于尚未确定的修饰因素的相互作用而获得存活的小鼠。由于很难预测从继续繁殖中获得存活时间更长的小鼠需要多长时间，我们也将使用替代策略。我们将通过培育高水平表达人类HbF的转基因来拯救镰状细胞小鼠，最好是在胎儿时期。一种策略是使用α-LCR(HS-40)结构来指导伽马珠蛋白基因的表达。此前，已有研究表明，当HS4-40被用于指导α-珠蛋白时，α-珠蛋白在胚胎中的表达很高，但在出生后不久就下降了。另一种选择是在贝塔嵌合转基因中繁殖，在这种转基因中，伽马珠蛋白基因的表达由贝塔启动子指导。挽救的转基因将在RNA和蛋白质水平上进行标记，以使它们的表达与YAC的表达有所不同。为了验证这一模型的实用性，将给镰刀细胞系注射已被发现影响人类伽马珠蛋白基因表达的药物。该模型还将用于其他研究人员测试增加胎儿血红蛋白或抑制血红蛋白S聚合的新药物。

This is a continuation of the effort to produce a mouse model of sickle anemia. During the past project years, we have succeeded in constructing a sickle cell mouse in which the predominant hemoglobin produced in the adult mouse is HbS. This model is different from others that have been made in the beta/S globin transgene lies within a YAC which contains the locus control region and the whole beta-globin gene cluster. Because the control regions of the globin genes and putative enhancers and silencers sequences are in their native context, these mice may mimic more closely to humans for testing drugs that affect HbF production. One problem in the preparation of these mice is that the yield is extremely low. This may be due to the fact that these mice turn off gamma-globin gene expression early in the embryo and the high level of HbS is not tolerated in utero. Our aim in the coming years is to brain sickle cell mice that survive the neonatal period using several approaches. First, we will continue to breed them in order to see if we can obtain more sickle cell mice that survive. It has been the experience of others that in spite of initial differences in obtaining sickle cell mice because of perinatal mortality, upon repeatedly breeding, mice that survive could eventually be obtained probably due to the interaction of yet undefined modifying factors. Because it is difficult to predict how long it will take to obtain longer surviving mice from continued breeding, we will also use alternative strategies. We will rescue the sickle cell mouse by breeding in transgenes that express high levels of human Hb F, preferably only in fetal life. One strategy is to use an alpha- LCR (HS-40) construct to direct gamma-globin gene expression. Previously, it has been shown that when the HS4-40 was used to direct an alpha-globin expression was high in the embryo but declined soon after birth. Another alternative is to breed in a beta-chimeric transgene in which the expression of the gamma-globin gene is directed by the beta promoter. The rescuing transgenes will be marked at both the RNA and protein levels to allow their expression to be differentiated from that of the YAC. To validate the utility of this model, the sickle cell line will be given agents that have been found to affect gamma-globin gene expression in humans. The model will also be available to other investigators to test new agents that increase fetal hemoglobin or inhibit hemoglobin S polymerization.