microscope. The male pronucleus is injected (still under the microscope) with STEP 1: Retrieve fertilized egg purified DNA fragments that mouse contain the bs gene along with an appropriate promoter region to s gene(into male pront give it a good chance of being expressed in once integrated into into fo the genome. The injected DNA quite often gets incorporated into the genome, and about one three into the ute eggs that are implanted into a About 10-30% of offspring foster mother mouse will have the ansgene integrated tissues and germ line Bs gene integrated and will go on to produce a baby mouse. Animals STEP 6: Breed transgenic in germ line that score positive for the human carriers of the transgene. transgene are mated to generate Figure by MIT OCW mice homozygous for the transgene. Among these progeny one is likely to contain the mutated human B-globin protein in its RBCs This was indeed achieved, bUt, this mouse did not prove to be a good model for sickle cell disease. It turns out that the human B-globin protein does Genotypes of the Bs H Transgenic Mice not complex well with the mouse a globin protein (a")and so the cloned gene encoding the human a- globin protein(a")was introduced Y-世 into fertilized mouse eggs to create a new transgenic mouse line which was Breed transgenic offsp then mated with the Bs transgenic mouse to produce a mouse expressing both Bs and a proteins Unfortunately the RBCs of these mice do not Note that the a gene is almost certain sickle efficiently.may be because human a- to integrate into different location than globin is not present the s gene did, and probably in a different chromosome These alleles will therefore sort independently when the two transgenic mouse lines are bred trdigeth n rea mbi together. The strong expectation was that the presence of the ah aBs Bs H hemoglobin tetramer in mouse RBCs would lead to the precipitation of fibersmicroscope. The male pronucleus is injected (still under the microscope) with purified DNA fragments that contain the βS H gene along with an appropriate promoter region to give it a good chance of being expressed in once integrated into the genome. The injected DNA quite often gets incorporated into the genome, and about one three eggs that are implanted into a foster mother mouse will have the βS H gene integrated, and will go on to produce a baby mouse. Animals that score positive for the human transgene are mated to generate mice homozygous for the transgene. Among these progeny one is likely to contain the mutated human β−globin protein in its RBCs. This was indeed achieved, BUT, this mouse did not prove to be a good model for sickle cell disease. It turns out that the human β-globin protein does not complex well with the mouse α- globin protein (αM) and so the cloned gene encoding the human α- globin protein (αH) was introduced into fertilized mouse eggs to create a new transgenic mouse line, which was then mated with the βS H transgenic mouse to produce a mouse expressing both βS H and αH human proteins. Genotypes of the βS H Transgenic Mice βM βM αM αM αM αM βM βM αM αM αM αM βH S βH S βH S Genotypes of the βS H Transgenic Mice βM βM αM αM αM αM βM βM αM αM αM αM βH S βH S βH S Note that the αH gene is almost certain to integrate into different location than the βS H gene did, and probably in a different chromosome. These alleles will therefore sort independently when the two transgenic mouse lines are bred together. The strong expectation was that the presence of the αH αH βS H βS H hemoglobin tetramer in mouse RBCs would lead to the precipitation of fibers Breed transgenic offspring Add in the human α-globin transgene and breed mice Add in the human α-globin transgene and breed mice Unfortunately the RBCs of these mice do not sickle efficiently……maybe because human α− globin is not present. βM βM αM αM αM αM βH S βH S βM βM αM αM αM αM βH S βH S αH αH Unfortunately the RBCs of these mice do not sickle efficiently……maybe because human α− globin is not present. βM βM αM αM αM αM βH S βH S βM βM αM αM αM αM βH S βH S αH αH βM βM αM αM αM αM βH S βH S αH αH STEP 1: Retrieve fertilized egg from recently mated female mouse. STEP 3: Human “transgene” integrates into the mouse genome at a random site. STEP 4: Transfer injected egg into the uterus of a foster mother. STEP 5: Foster mother gives birth to pups, about 1 in three have the transgene integrated into every cell of its body. STEP 6: Breed transgenic offspring to get homozygous carriers of the transgene. STEP 2: Inject cloned human βS H gene (into male pronucleus). Transfer injected eggs into foster mother Inject foreign DNA into one of the pronuclei Fertilized mouse egg prior to fusion of male and female pronuclei Pronuclei About 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line Breed mice expressing foreign DNA to propagate DNA in germ line Figure by MIT OCW