Restriction Fragment Length Polymorphism Combined DNA Index System

Restriction Fragment Length Polymorphism

Combined DNA Index System

Methods for inserting the recombinant DNA into a cell: • Cells may be treated with chemicals to make plasma membranes more permeable—DNA diffuses in. • Electroporation—a short electric shock creates temporary pores in membranes, and DNA can enter. • Viruses and bacteria can be altered to carry recombinant DNA into cells. • Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs. • “Gene guns” can “shoot” the host cells with particles of DNA.

• Selectable markers are a type of reporter gene—a gene whose expression is easily observed. • Green fluorescent protein, which normally occurs in a jellyfish, emits visible light when exposed to UV light. • The gene for this protein has been isolated and incorporated into vectors as a reporter gene.

GFP

A team of researchers, led by Eric Poeschla of the Mayo Clinic College of Medicine, have come up with an adorable helper in the push to find a cure for HIV/AIDS: the kitten who glows green under UV light. This green-glowing kitten has had genetic material, green fluorescent protein to be exact, from the jellyfish Aequorea victoria fused with its own. So how does the green fluorescent protein help scientists in their research to find a cure? It doesn’t directly, but it acts as a tracking mechanism that allows researchers to know that the kitten’s genetic material was successfully infused with not only the green-glow-gene, but also a gene called TRIMCyp found in HIV-resistant rhesus macaque monkeys. Researchers hope that by infusing this genetic material with the cat’s own, they will create a “naturally” immunity to Feline Immunodeficiency Virus (FIV), which, like HIV in humans, causes AIDS in cats. HIV and FIV strip the immune system’s T-cells, necessary for fighting off infections (remember, AIDS isn’t the actual cause of death – it is usually caused by some minor infection, like the common cold, that is allowed to run rampant because the immune system is completely disabled). There are key proteins called restriction factors that defend against viruses, but ours and cats’ can’t stand up to HIV/FIV. The rhesus macaque monkey has a restriction factor, TRIMCyp, that is capable of attacking and disabling the HIV/FIV’s outer shield as it tries to invade a cell. The research team has taken white blood cells from the genetically-altered cats, exposed them to FIV and found that they are immune.

Figure 1. The Sanger sequencing reaction. Single stranded DNA is amplified in the presence of fluorescently labelled dd. NTPs that serve to terminate the reaction and label all the fragments of DNA produced. The fragments of DNA are then separated via polyacrylamide gel electrophoresis and the sequence read using a laser beam and computer.