Cells from the inner cells mass of a preimplantation embryo at the blastocyst stage could be removed and cultured in the lab without the cells losing their totipotency i e. even after being cultured in the lab for many years these cells can still be introduced back into a preimplantation embryo and go on to make all the tissues of a mouse. What this means, is that the cells can be genetically manipulated whilst in culture. and then put back into a mouse preimplantation embryo Preimplantation blastocyst from an th gpewoduce a mouse (aor B-globin genes) EMBRYONIC STEM CELLS and ganciclovir (ES cells)and culture in vitro Formation of ES Cells Carrying a Knockout Mutation Es cells (originally from a blastocyst for a and against the mew elasto st that ouinnonnatyntove rise to a mouse with wHITE FUR ed into a foster mother: she will give birth to the chimeric offspring Select fot the genetically Figures by MIT OCW Essentially once you have identified mouse es cells originally from a grey furred mouse) that have been genetically altered the way you wish . these cells can be used to generate a living animal that contains descendents from these totipotent Es cells. Lets see how you get from there to a mouse in which every cell contains that genetic alterationCells from the inner cells mass of a preimplantation embryo at the blastocyst stage could be removed and cultured in the lab without the cells losing their totipotency; i.e., even after being cultured in the lab for many years these cells can still be introduced back into a preimplantation embryo and go on to make all the tissues of a mouse. What this means, is that the cells can be genetically manipulated whilst in culture…and then put back into a mouse preimplantation embryo!! Essentially, once you have identified mouse ES cells (originally from a grey furred mouse) that have been genetically altered the way you wish…these cells can be used to generate a living animal that contains descendents from these totipotent ES cells. Lets see how you get from there to a mouse in which every cell contains that genetic alteration. Preimplantation blastocyst from an embryo that would produce a mouse with GREY FUR Can remove totipotent EMBRYONIC STEM CELLS (ES cells) and culture in vitro Select for the genetically altered cells you want Construct Specifically replace your gene of interest (α or β-globin genes) with a mutated version of that gene in cultured ES cells Gene X replacement construct neor tkHSV Homologous recombination Gene-targeted insertion Gene X Mutation in gene X Cells are resistant to G-418 and ganciclovir Formation of ES Cells Carrying a Knockout Mutation ES cells ES-cell DNA Random insertion Other genes No mutation in gene X Cells are resistant to G-418 but sensitive to ganciclovir ES cells ES-cell DNA Nonhomologous recombination Targeting Construct Select for the NeoR gene and against the TKHSV gene The only cells to survive have undergone a targeted homologous recombination event at the gene of interest Select fot the genetically altered cells you want NeoR TKHSV Now you inject the genetically modified ES cells (originally from a blastocyst for a mouse with GREY FUR) and inject into a new blastocyst that would normally give rise to a mouse with WHITE FUR The blastocyst, now containing two types of totipotent embryonic stem cells, is implanted into a foster mother; she will give birth to the chimeric offspring Figures by MIT OCW