Slide 1: Introduction
Introduce myself (Name, sophomore, Knockout Mice)
Slide 2: (what is a knockout mouse)
~A knockout mouse is a genetically engineered mouse.
~Researchers use the technique by “knocking out”/replacing the gene with an artificial piece of DNA. ~They do this so they could determine the specific function of the missing gene. ~Since humans are closely similar to mice, they can perform experiments on knockout mice and see the effects of the gene’s loss. ~This gives us a better understanding of a specific disease. ~This is a picture of a laboratory mouse where a gene is affecting hair growth had been knocked out (left), is shown next to a normal lab mouse.
Slide 3: (history)
~Mario R. Capecchi, Martin Evans and Oliver Smithies created the first known knockout mouse. ~They identified and separated the embryonic stem cells of the early embryo, the cell from which all cells of the adult organism are derived. ~He then established it in cell culture, modified it genetically and reintroduced it into foster mothers to generate a genetically modified offspring. ~In the picture (starting from the left, Oliver Smithies, Martin J. Evans and Mario R. Capecchi.
~Making a knockout mouse involves several steps. Takes about 2 yrs to get a mouse.
~In the first step of creating a knockout mouse, a “knockout” DNA construct is introduced into donor embryonic stem cells by electroporation. ~This construct is designed to introduce antibiotic resistance to cells if it gets in and recombines with the gene of interest.
~This is a picture of mouse embryonic stem cells growing on a bed of fibroblasts. These cells are put into a cuvette along with the DNA construct and loaded into the electroporator. ~This is an electroporator. It has a place for the cuvette to sit. The cells and DNA construct are then exposed to electricity and this changes the permeability of the cell membrane and DNA goes into the cells.
Next, the cells undergo homologous recombination
Slide 7: Stage 2 homologous recombination
~Once the knockout DNA construct is in the stem cell, it recombines with the original gene and deletes it from the genome. It replaces the original gene with the knockout construct conferring antibiotic resistance.
~For some unknown reason, not all stem cells receive the construct, thus you have to select the cells that received the construct.
Slide 9: Stage 3 Selection
~The knockout DNA construct contains the neomycin resistance gene. So if you plate the electroporated cells onto neomycin-containing plates, only cells containing the knockout construct will survive. All the others will die.
~You then take the surviving cells and inject them into a blastocyst.
Slide 11: Pronuclear injection
~Selected stem cells containing the knockout construct are then injected into the blastocyst of a recipient, wild type female mouse. ~Here, you can see the recipient blastocyst being held still by a device on the left. And here is the needle introducing the selected stem cells into the recipient blastocyst. This blastocyst now contains two types of stem cells, the original from the recipient mouse and the knockout stem cells from the donor.
~The babies are allowed to develop.
~The newborns would become chimeras.
~Chimeras are single celled organism that has both original stem cells and knocked out stem cells.
Slide 14: Possibilities
~If it is Germline, the recombinant stem cell becomes the reproductive cells, then all of the offspring would be Knocked out mice. ~If the construct wasn’t in the reproductive cells, it is non-germline, only some tissues and organs are knocked out.
Slide 15: Lethality
~ About 15% of gene knockouts are developmentally lethal, meaning they are incapable of growing into adult mice. ~ The insufficient number of adult mice limits studies to embryonic development. This makes it difficult to determine a gene’s function in relation to humans’ health. ~ In some cases, the gene of an adult mice and a developing embryo may vary drastically.