Molecular genetic techniques for gene manipulation in Candida albicans

Figures & data

Figure 1. Cassettes used for gene manipulation in C. albicans. The figure shows the necessary elements in these cassettes, the light blue color of the elements represents the 5′ and 3′ DNA flanking your target gene (YTG) or the upstream and downstream sequences of YTG. (1) URA blaster cassette.²⁴ (2) PCR amplifiable marker cassettes from non-C. albicans Candida species.⁴⁵C.d.HIS1, Candida dubliniensis HIS1; C.m.LEU2, Candida maltosa LEU2; C.d.ARG4, Candida dubliniensis ARG4. (3) URA flipper cassette.³⁸ prom, promoter region given for gene; T, termination sequence of given gene. (4) Cre-loxP system.⁴⁶ (5) MPA^R flipper cassette.⁴⁷ (6) SAT1 flipper cassette.⁵¹C.a.SAT1, C. albicans SAT1. (7) “Tet-Off” system.²⁰ (8) “Tet-On”system.⁵⁴ (9) Promoter exchange cassette.^18,60 (10) Cassettes used for gene reintegration.³⁴C.a.SAT1, C. albicans SAT1; C.a.LEU2, C. albicans LEU2; C.d.ARG4, C. dubliniensis. (11) ACT1P-GFP-ACT1T cassette.⁷⁶ (12) The firefly luciferase selectable marker cassette.⁷⁷ (13) PCR-mediated gene-tagging cassette.¹⁶ (14) Epitope tagging cassette.²³

Table 1. Cassettes used for gene manipulation in C. albicans

Cassettes	Purposes	Parent strains	Selective markers	Application examples	References
URA blaster cassette	Gene disruption	CAI4	URA3	Disrupting gene in auxotrophic strains	24
URA flipper cassette	Gene disruption	CAI4	URA3	Disrupting gene in auxotrophic strains	38
PCR amplifiable URA3 cassette	Gene disruption	RM1000, BWP17	URA3	Disrupting gene in auxotrophic strains	19
UAU1 cassette	Gene disruption	BWP17	URA3, ARG4	Disrupting gene in auxotrophic strains	43
PCR amplifiable marker cassettes from non-C. albicans Candida species	Gene disruption	SN87, SN95, SN152	HIS1, LEU2, ARG4	Disrupting gene in auxotrophic strains	45
Cre-loxP system	Gene disruption	BWP17	HIS1, URA3, ARG4	Disrupting gene in auxotrophic strains	46
MPA^R flipper cassette	Gene disruption	Any strain	IMH3	Disrupting gene in any strains	47
SAT1 flipper cassette	Gene disruption	Any strain	SAT1	Disrupting gene in any strains	51
“Tet-Off” system	Controlled gene expression	CaSS1	SAT1, URA3	Repressing the expression of the target gene	20
“Tet-On”system	Controlled gene expression	CAI4	SAT1,URA3	Inducing the normally not expressed gene or inducing the deletion of essential gene	54
Promoter exchange cassette	Controlled gene expression	BWP17, SN148, SN152	URA3, HIS1, ARG4, LEU2, SAT1	Repressing the expression of target gene	18 and 60
PCR-directed recombination in S. cerevisiae	Gene reintegration and overexpression	SN152, SN147	ARG4, SAT1	Inducing overexpression of target gene	65
ACT1P-GFP-ACT1cassette	Protein tagging	CAI4	URA3	To locate the target protein	76,110, and 111
The firefly luciferase selectable marker cassette	Protein tagging	CAI4	URA3	To locate the target protein	77 and 78
PCR-mediated gene-tagging cassette	Protein tagging	CAI4, NGY152	SAT1	To locate the target protein	16
Epitope tagging for antibody detection	Protein tagging	BWP17, SN76	HIS1, URA3, ARG4	To analyze the protein complex	23

Table 2. The commonly used auxotrophic strains in gene manipulation in C. albicans

Auxotrophic strains	Phenotype	Features	References
CAI4	Ura^−	Decreased virulence; having chromosome 2 trisomy	24, 29, 32, 33, 63, 80, 81, and 86
RM1000	His^−, Ura^−	Decreased virulence; lacking the right arm of chromosome 5	25, 26, 29, 32, 33, 63, 80, 81, and 86
BWP17	Arg^−, His^−, Ura^−	Decreased virulence; lacking the right arm of chromosome 5	25, 26, 29, 32, 33, 63, 80, 81, and 86
CaSS1	His^−, Ura^−	Decreased virulence	20, 29, and 33
NGY152	Ura^−	Full virulence	16 and 29
SN76	Arg^−, His^−, Ura^−	Decreased virulence; having no chromosome change	23, 45, and 87
SN87	Leu^−, His^−	Full virulence	45
SN95	Arg^−, His^−	Full virulence; having no chromosome change	45 and 87
SN100	His^−	Full virulence	45
SN148	Arg^−, Leu^−, His^−, Ura^−	Decreased virulence; having no chromosome change	18, 45, 60, and 87
SN152	Arg^−, Leu^−, His^−	Mild virulence defect; having no chromosome change	18, 45, 60, and 87

Figure 2.URA blaster strategy used for gene disruption in C. albicans.³⁹ YTG, your target gene.

Figure 3. Strategy of the fusion PCR and heterologous markers used for gene disruption in C. albicans.⁴⁵ YTG, your target gene. C. d, Candida dubliniensis; C. m., Candida maltosa.

Table 3. The commonly used regulatable promoters in gene manipulation in C. albicans

Cassettes	Promoters	Inducer	Gene expression	References
“Tet-On”system	TET	Doxycycline	Induced	55
“Tet-Off”system	TET	Tetracycline	Repressed	20
Marker gene-MET3p	MET3	Methionine, cysteine	Repressed	18, 59, and 60
Marker gene-MAL2p	MAL2	Glucose	Repressed	18, 59, and 60
Marker gene-MAL2p	MAL2	Maltose	Induced	18, 59, and 60
G1G1p-GFP cassette	G1G1	GlcNAc	Induced	61
URA flipper cassette	SAP2	YCB-BSA pH 4.0	Induced	38 and 47–49
MPA^R flipper cassette
SAT1 flipper cassette	MAL2	Maltose or glucose	Induced	51–53
Cre-loxP system	MET3	Methionine, cysteine	Repressed	46

Figure 4. Strategy of the Cre-loxP system used for gene disruption in C. albicans.⁴⁶ YTG, your target gene; prom, promoter region given for gene.

Figure 5. The SAT1 flipper strategy used for gene disruption in C. albicans.⁵¹ YTG, your target gene; prom, promoter region given for gene; T, termination sequence of given gene.

Figure 6. Strategy of the “Tet-Off” system used to repress the expression of the target gene in the presence of tetracycline.²⁰ YTG, your target gene; prom, promoter region given for gene; T, termination sequence of given gene; BC1, up tag; BC2, down tag.

Figure 7. Strategy of the “Tet-On” system used to induce the deletion of essential genes.⁵⁴ YTG, your target gene; prom, promoter region given for gene; T, termination sequence of given gene.

Table 4. The commonly used tagging in C. albicans

Epitope tagging for antibody detection		Fluorescent markers
Tagging	Maker genes	Tagging	Maker genes
HA	URA3, HIS1, ARG4	GFP	URA3, CaHIS1, CmHIS1, ARG4, CmLEU2, SAT1
MYC	URA3, HIS1, ARG4	YFP	SAT1, URA3
TAP	URA3, HIS1, ARG4	CFP	SAT1, URA3
GST	URA3	RFP	SAT1, URA3
His9	URA3	luc	URA3
V5–6 × His	URA3, SAT1

Figure 8. Strategy of protein tagging at the C-terminus of the target gene used in C. albicans.¹⁶ YTG, your target gene; prom, promoter region given for gene; T, termination sequence of given gene.

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