Fig. 3.

Conditional knockout of the TbRab7 locus in PCF trypanosomes.

A. Using flanking primers the TbRab7 locus was PCR amplified from PCF wild type (WT), TbRab7 single knockout (1KO) and TbRab7 conditional double knockout (cKO) genomic DNA. The HSP70 orf was also amplified as a positive control for amplification and loading. PCR products were fractionated on 0.8% agarose gels. Sizes of PCR products are indicated (nts). Note that PCR products of the first round (2060 nts) and second round (1860 nts) allelic replacement constructs are close in size.

B. Total RNA (10 μg/lane) from the PCF cKO cell line grown in the absence (−) and presence (+) of tetracycline for 6 days was probed for TbRab7 mRNA as in Fig. 2. The blot was stripped and reprobed for tubulin as a loading control.

C. Growth curves of the PCF cKO cell line in the absence and presence of tetracycline. Cultures were adjusted to starting cell density every 3 days. Data are presented as means ± SEM (n = 3).