Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

Table 1 Frequencies of Cre-mediated insertion events

^a Host cell	Insertion construct	^b Cre	Drug Selection	^c Colonies	^d GFP-neg	^e Tet	^f PCR/SB	^g I
Rht14-19	pIN-1	+	hyg	53	47/48	47/48	10/10	10
Rht14-19	pIN-1	-	hyg	0	-	-	-	0
Rht14-10	pIN-2	+	MPA/X	11	9/11	9/11	9/9	1.8
Rht14-10	pIN-2	-	MPA/X	0	-	-	-	0
Rht14-19	pIN-2	+	MPA/X	32	22/33	22/33	9/9	4.4
Rht14-19	pIN-2	-	MPA/X	0	-	-	-	0
Rht14-10	pIN2-neoMCS	+	G418	188	181/188	NT	NT	36
Rht14-10	pIN2-neoSCE	+	G418	169	163/169	3/3	3/3	33
Rht14-10	pIN2-neoR52	+	G418	226	217/226	3/3	3/3	43

^a Each row represents data for the transfection of 5 × 10⁵ cells.
^b Indicates whether pMC-Cre was (+) or was not (-) co-transfected.
^c Total number of drug-resistant colonies generated.
^d Colonies that did not express GFP.
^e GFP-negative colonies that became drug-sensitive on addition of tet.
^f GFP-neg/Tet^s colonies with expected structure detected by PCR or Southern Blot.
^g Cre-mediated insertion frequency expressed as events per transfected cell × 10⁵ (assuming all GFP-neg clones are correctly inserted).

ISSN: 1471-2199