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Generation of CFAS mutant parasites and in vitro growth analysis.

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posted on 2012-12-10, 02:30 authored by Samuel O. Oyola, Krystal J. Evans, Terry K. Smith, Barbara A. Smith, James D. Hilley, Jeremy C. Mottram, Paul M. Kaye, Deborah F. Smith

(A) Region of chromosome 8 containing the single L. infantum CFAS locus and the constructs used for targeted gene deletion. (B) Representative Southern blot of L. infantum and L. major wild type (wt) and CFAS mutant DNAs hybridised with a CFAS-specific probe (see A above and Materials and Methods). Two independent L. infantum CFAS null clones (1 and 2, −/−) are shown; the single allele deletion prior to generation of null clone 2 (+/−) and a complemented add-back clone from that line (−/−/+, CLN-C2, Table 1) are included. One of the clones of L. major transgenic for luciferase and CFAS (+CFAS, CLN-4, Table 1) is also shown. A labelled β-tubulin specific DNA probe was used as a loading control. (C) In vitro growth of L. infantum cell lines. L. infantum wild type and CFAS null and complemented lines (as analysed in (B)) were grown over 5 days in HOMEM/20% FCS at 26°C and parasites counted as described (Materials and Methods). Mean values derived from triplicate culture populations for each cell line are plotted. (D) In vitro growth of L. major cell lines. L. major wild type containing an integrated luciferase gene (L. major LUC) and the same line expressing CFAS (analysed in B) were grown over 7 days in either M199/20% FCS (top) or HOMEM/20% FCS (bottom) and parasites counted as described in (C).

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