It is the aaBsBs hemoglobin molecule expressed ges removed due after birth that is responsible for aggregating Sickle Cell Trait: aaBB. to copyright reasons. and causing sickle cell disease. The aapBs hemoglobin tetramers expressed in people heterozygous for the sickle mutation do not Sickle Cell Disease: aaB. B aggregate to form fibers and so do not cause disease; however, should such heterozygous apsS is soluble when oxygenated, but precipitates people live at high altitude some sickling can occur It is sobering to note that almost 50 years since the molecular basis of this disease was discovered there still does not exist a really effective therapy for the disease. Hemoglobin was one of first proteins to be purified it's gene was one of the first to be cloned and the globin proteins were among the first to have their structure determined by x-ray crystallography. and although some progress has been made in therapy much more still needs to be done. this is precisely why having a robust mouse model for sickle cell disease to test experimental therapies is absolutely critical. Tremendous strides have been made in generating a mouse model for sickle cell disease How do we Genetically modify There are two general ways to specifically the mouse genome modify the genetic makeup of a mouse. one nvolves the random integration of a cloned gene somewhere into the mouse genome (i.e random insertion with no replacement the introduction of a transgene). The other involves precisely targeting a specific gene in (2)“ Knock-outs the mouse and introducing a know alteration of subtracting or deleting genes gene targeting that gene, usually the deletion of the gene and pecific insertion with replacement the insertion of a marker gene in its place(a gene knock-out by targeted homologous recombination) Introduction of the Human B-globin gene with the sickle cell mutation (Bs)into the mouse genome: In the 1980s and early 1990s several groups tried to make a mouse with sickle cell disease by introducing the Human B-globin gene with the sickle mutation(Bs ) in the hope that if the protein was expressed at high levels it would precipitate Hb fibers that would make a transgenic mouse? mimicking sickle cell disease. How does one cause sickling of rbcs thus Mice are treated with a hormone to make them super-ovulate and then mated Soon after mating the fertilized eggs are retrieved from the uterus. Eggs that contain two pronuclei (one from the mother and one from the father) indicating that the embryo is still at the one-cell stage, are identified under theIt is the ααβSβS hemoglobin molecule expressed after birth that is responsible for aggregating and causing sickle cell disease. The ααββS hemoglobin tetramers expressed in people heterozygous for the sickle mutation do not aggregate to form fibers, and so do not cause disease; however, should such heterozygous people live at high altitude some sickling can occur. Healthy people: ααββ Sickle Cell Trait: ααββs Sickle Cell Disease: ααβsβs ααβsβs is soluble when oxygenated, but precipitates in low oxygen Healthy people: ααββ Sickle Cell Trait: ααββs Sickle Cell Disease: ααβsβs ααβsβs is soluble when oxygenated, but precipitates in low oxygen It is sobering to note that almost 50 years since the molecular basis of this disease was discovered there still does not exist a really effective therapy for the disease. Hemoglobin was one of first proteins to be purified, it’s gene was one of the first to be cloned, and the globin proteins were among the first to have their structure determined by x-ray crystallography…and although some progress has been made in therapy, much more still needs to be done. This is precisely why having a robust mouse model for sickle cell disease to test experimental therapies is absolutely critical. Tremendous strides have been made in generating a mouse model for sickle cell disease. There are two general ways to specifically modify the genetic makeup of a mouse. One involves the random integration of a cloned gene somewhere into the mouse genome (i.e., the introduction of a “transgene”). The other involves precisely targeting a specific gene in the mouse and introducing a know alteration of that gene, usually the deletion of the gene and the insertion of a marker gene in its place (a gene knock-out by targeted homologous recombination). How do we Genetically modify the mouse genome? (1) Transgenes (2) “Knock-outs” • adding genes by pronuclear injection • random insertion with no replacement • subtracting or deleting genes • gene targeting • specific insertion with replacement How do we Genetically modify the mouse genome? (1) Transgenes (2) “Knock-outs” • adding genes by pronuclear injection • random insertion with no replacement • subtracting or deleting genes • gene targeting • specific insertion with replacement Introduction of the Human β-globin gene with the sickle cell mutation (βS H) into the mouse genome: In the 1980’s and early 1990’s several groups tried to make a mouse with sickle cell disease by introducing the Human β-globin gene with the sickle mutation (βS H), in the hope that if the protein was expressed at high levels it would precipitate Hb fibers that would cause sickling of RBCs, thus mimicking sickle cell disease. How does one make a transgenic mouse? Mice are treated with a hormone to make them super-ovulate and then mated. Soon after mating, the fertilized eggs are retrieved from the uterus. Eggs that contain two pronuclei (one from the mother and one from the father) indicating that the embryo is still at the one-cell stage, are identified under the Images removed due to copyright reasons