Table 1.

Summary of approaches for analysis of erythrocyte determinants of 698 Plasmodium infection.

Approach	Pros	Cons
Natural Mutants
Assays using natural erythrocyte mutants	Polymorphisms selected by nature (physiologically relevant) Viable erythrocytes naturally obtained	Confounding polymorphisms also present Difficult to control experiments with cells from different donors Inability to complement the mutation
Functional genomics of natural mutants	Using natural selection to identify novel candidate resistance/susceptibility loci	Weak signals due to (weak) linkage disequilibrium To date, GWAS has required further deep sequencing to uncover signals
Biochemical Approaches
Pull-down	Detects physiologically interacting proteins, ex vivo or in vitro, with or without cross-linkers	Possible that weak interactions will not be detected by pull-down
Gel-overlay, Erythrocyte binding assays (EBAs) with enzyme treated erythrocytes	Can use both antibody mediated detection or radioactive methods	Protein abundance and stability can effect the sensitivity Requires stable protein interactions
Invasion assays with enzyme treated erythrocytes	Determine the effect of removing sets of receptors in the same genetic background on parasite invasion	Non-specific removal of receptors. Removes groups of both known and unknown receptors.
Antibody mediated inhibition	Specific inhibition of a receptor by blocking access to the parasite ligands Can determine the effect of blocking a given receptor in the same genetic background High throughput	Antibody dependent (not all antibodies are inhibitory) Agglutination Off target effects of antibodies (can impact both invasion and membrane rigidity, for example)
Avexis	Pentamerizing the bait or prey increases detection of transient interactions	Limited by what can be expressed (size, glycosylation, number of transmembrane domains) False positives
Chemical genetic Approaches
Chemical inhibition	Potent inhibition can be achieved Temporal inhibition can be achieved Depending on the inhibitor, inhibition can be reversed	Off-target effects Specificity of inhibition can be dependent on concentration Host versus parasite specificity can be a narrow window, and difficult to achieve
Immunodepletion of cytoplasmic proteins	The alternative to transgenic modification with the same result – specific depletion of a given protein	Limited to cytoplasmic proteins
Transgenic Approaches
shRNA Knock-down	Stable and either inducible or constant Amenable to forward genetic screens Can study the effect of the absence (or decrease) of a given protein in the erythrocyte	Level of knock-down varies depending on the gene Knock-down (rather than knock-out) can be insufficient to yield a phenotype Erythrocyte viability can be effected depending on when the knock-down has an effect May be necessary to induce knock-down later in erythropoiesis to avoid developmental effects
Ectopic expression	Inducible expression, varying level of expression, can be achieved with specific vectors and promoters	Matching physiologic levels of expression with appropriate promoters
Allelic replacement	Complementation is possible (ectopically expressing a protein in a null background)	Must codon optimize the transgenically expressed allele to avoid shRNA mediated KD of replacement as well as endogenous Matching levels of expression with appropriate promoters

GWAS, genome-wide association study; shRNA, small hairpin RNA; KD, knock down; AVEXIS, avidity-based extracellular interaction screen.