FIG 3.

KSHV ORF57 KO in BCBL-1 cells. (A) Work flow showing multiple rounds of BCBL-1 cell transfection with the second generation of gRNA vectors followed by 3 days of puromycin selection. After achieving a detectable level of ORF57 KO, the cells were subjected to prolong puromycin selection and single-cell cloning by limited dilution in a 96-well plate. The obtained clones were expanded and used for PCR screen. (B) The KSHV ORF56-ORF57 locus, targeted sites by gRNAs (red squares), and PCR screening primers (black arrows below the diagram). (C) The efficiency of ORF57 KO in BCBL-1 cells after 1, 2, and 4 selection cycles as determined by PCR using Pr1 plus Pr2 primers. (D and E) ORF57 KO and PCR screening of isolated BCBL-1 single-cell clones derived from gRNA 2 plus 4 vector stable transfection by using Pr1 plus Pr2 (D) or Pr1 plus Pr3 (E) primers as described in panel B. WT, wild type ORF57 gene product; KO, knockout ORF57 gene product; B-1, parental BCBL-1 cell population.