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Retrovirus Infection Protocols

Containing an RNA genome that is encapsulated in a capsid and lipid envelope, retroviruses contain polypeptide chains that help link them to receptors in the membranes of host cells they infect. Whereas most cells' hereditary information is coded in DNA, that of retroviruses is contained in RNA; retroviruses also carry the enzyme reverse transcriptase, allowing them to adhere to the host DNA. Certain protocols are followed by laboratories in the purposeful infection of the DNA cell with these viruses.

  1. Preparing the Laboratory

    • Certain laboratory equipment and supplies are necessary to aid in the process of retroviral infection. Equipment includes 1 pipette aid, 1 pipette-man, 1 tissue culture hood, 37-degree Celsius and 32-degree Celsius incubators, and an Eppendorf centrifuge. Supplies include tissue culture plates, a disposable Pasteur pipette, qualified retroviral supernatant, filter-sterilized polybrene and filter-sterilized protamine sulfate.

    Preparing the Cell for Infection

    • Prior to infection, target cells must be split in a tissue-culture plate and incubated overnight in a 37-degree Celsius incubator with five percent CO2. The purpose of the incubation period is to create cells which are 50% confluent. Preparing the cells beforehand is necessary to ensure that the cells are optimized for the infection process. The Nolan Lab at Stanford University recommends that cell infection always begin start by infecting the NIH 3T3 cells to help optimize the results of the test.

    Infecting the Cells

    • After the incubation period is over, researchers should ensure that the cells are both healthy and of the right confluence. Using the tissue-culture hood, the cellular matter is aspirated and a viral supernatant is applied to the cells. Once applied, the plate should be gently rocked back and forth to ensure that it is evenly distributed. Cells should then be spin-inoculated by placing them in the Eppendorf centrifuge and spinning them at 2,500 rpm for ninety minutes at a temperature of 30 degrees Celsius. Afterward, the cells should be incubated at 32 degrees Celsius for one to two days. The virus is then replaced with the extracted cells and placed into the incubator to be analyzed anywhere from three to seven days after the initial infection.

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