1.In Figure 1b why do only pluripotent cells survive? RETROVIRUSES CAN INSERT THEMSELVES RANDOMLY INTO THE GENOME AND CAUSE CELLS TO SPONTANEOUSLY ACQUIRE PLURIPOTENT CHARACTERISTICS WHICH LIKE CANCER CAUSE THEM TO OVERGROW. FBX15 IS ONLY EXPRESSED IN PLURIPOTENT CELLS SO ONLY PLURIPOTENT CELLS WILL HAVE GEOMYCIN RESISTANCE AND SURVIVE ANTIBIOTIC TREATMENT. G418 IS A NATURAL INHIBITOR OF NON-PLURIPOTENT CELLS. 2.In Figure 2a how do the authors decide to pursue their top 10 candidates? THE NUMBER OF COLONIES FORMED IS INVERSELY PROPORTIONAL TO THE DEGREE OF RETROVIRAL INTEGRATION. ANY FACTOR THAT HAD MORE THAN 20 COLONIES WAS SELECTED. REMOVING THE FACTORS MOST CRITICAL TO REPROGRAMMING SHOULD LEAD TO LOSS OF COLONY FORMATION. EFFECTIVE VIRAL TRANSDUCTION CAN BE READ OUT USING A GENETIC REPORTER. 3.In Figure 3a why is it important to not detect transgenic transcripts? THE RETROVIRUS IS INTEGRATED AT THE SAME LOCUS EACH TIME AND THUS LEADS TO VERY STABLE GENE EXPRESSION. IT IS ONLY POSSIBLE TO MEASURE ENDOGENOUS TRANSCRIPTS BY RT-PCR. TRANSGENES ARE RANDOMLY INTEGRATED AND KNOCKINS ARE SPECIFICALLY INTEGRATED. 4.TRUE OR FALSE c-myc protein is expressed in MEFs 5. 5.In Figure 7c the authors are examining integration of the retroviruses in many different iPSC lines. What are they looking at here? DIFFERENT KLF4 HOMOLOGUES IN DIFFERENT IPSC LINES. THE EXPRESSION OF KLF4 MRNA IN IPSCS. THE LOCATION OF KLF4 CDNA IN THE GENOMIC DNA. KLF4 PROTEIN EXPRESSION. Requirements: all questions ‘
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