QUICK SEARCH:

[advanced]

	Author:		Keyword(s):

Home Help Feedback Subscriptions Archive Search Table of Contents

	Help viewing high resolution images
	Return to article

(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.

Fig. 3. Generation of metacaspase null mutants. (A) Schematic representation of the MCA2-MCA3 and MCA5 loci together with constructs for gene knockout. Open reading frames (ORFs) are shown as arrows, flanking DNA sequences for targeting are shown as boxes. The predicted fragment sizes of KpnI-digested DNA for both native and modified MCA2-MCA3 locus is shown. BSD, blasticidin-resistance gene; PAC, puromycin-resistance gene; HYG, hygromycin-resistance gene; NEO, neomycin-resistance gene. (B) Southern blot analysis of ${Delta}$ mca2/3. Genomic DNA was digested with KpnI, separated on a 1% agarose gel, blotted onto Hybond-P membrane and hybridised with a ³²P-labelled DNA probe comprising the 5' flanking region used in the knockout strategy. Lane 1, wild type; lane 2, PAC heterozygote; lane 3, BSD heterozygote; lane 4, ${Delta}$ mca2/3 clone 1; lane 5, ${Delta}$ mca2/3 clone 2. (C) Total cell lysates were prepared from BSF parasites and 5x10⁶ cell equivalents subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with rabbit anti-MCA2-MCA3 antibodies. Lane 1, wild type; lane 2, ${Delta}$ mca2/3; lane 3, ${Delta}$ mca2/3 ${Delta}$ mca5; lane 4, ${Delta}$ mca2/3 ${Delta}$ mca5:MCA3; lane 5, ${Delta}$ mca2/3 ${Delta}$ mca5:MCA2. MCA2 and MCA3 are indicated. Asterisk indicates crossreacting 48 kDa protein. (D) Total cell lysates were prepared from BSF parasites and 5x10⁶ cell equivalents subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with sheep anti-MCA5 antiserum. Lane 1, wild type; lane 2, ${Delta}$ mca5; lane 3, ${Delta}$ mca2/3 ${Delta}$ mca5; lane 4, ${Delta}$ mca2/3 ${Delta}$ mca5:MCA5. MCA5 is indicated.

Return to article