SICKLE CELL ANEMIA MOUSE MODEL

这是一个继续的努力，以产生一个小鼠模型的镰状贫血。在过去的几年里，我们已经成功地构建了镰状细胞小鼠，其中成年小鼠中产生的主要血红蛋白是HbS。该模型与其他模型不同，β/S球蛋白转基因位于包含基因座控制区和整个β球蛋白基因簇的YAC内。由于球蛋白基因的控制区和假定的增强子和沉默子序列处于其天然环境中，这些小鼠可以更接近于人类来测试影响HbF产生的药物。在制备这些小鼠时的一个问题是产量极低。这可能是由于这些小鼠在胚胎早期关闭了γ-珠蛋白基因表达，并且在子宫内不能耐受高水平的HbS。我们在未来几年的目标是使用几种方法使镰状细胞小鼠在新生儿期存活下来。首先，我们将继续繁殖它们，看看我们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率而在获得镰状细胞小鼠方面存在初始差异，但在反复繁殖后，最终可能由于尚未确定的修饰因子的相互作用而获得存活的小鼠。 因为很难预测需要多长时间才能从继续繁殖中获得存活时间更长的小鼠，所以我们也将使用替代策略。我们将拯救镰状细胞小鼠繁殖的转基因表达高水平的人类血红蛋白F，最好只在胎儿期。一种策略是使用α- LCR（HS-40）构建体来指导γ-珠蛋白基因表达。以前，已经表明，当HS 4 -40用于指导α-珠蛋白表达时，胚胎中的α-珠蛋白表达很高，但出生后很快下降。另一种选择是在β-嵌合转基因中繁殖，其中γ-珠蛋白基因的表达由β启动子指导。拯救转基因将在RNA和蛋白质水平上被标记，以使它们的表达与YAC的表达区分开。为了验证该模型的实用性，镰状细胞系将被给予已经发现影响人类γ-珠蛋白基因表达的药剂。该模型也将提供给其他研究人员，以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.