A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones

Table 2 Resources for simplified counter-selection recombineering

			RT-cassette-containing construct^b		Replacement construct^c
Desired insertion sequence^a	RT	kb^d	Fwd (5^′-3^′)^e	Rev (5^′-3^′)^e	pCC1Fos-based	pGOv5-based	kb^f
RT-cassette	pNH034	2.0	GCTGTCGAGATATGACGGTGTTCA	TCTTGGAGTGGTGAATCCGTTAGC	-	-
F-CFP	pNH050	2.4	ATGAGTAAAGGAGAAGAACTTTTC	[*]TTTGTATAGTTCATCCATGCCATG	pNH039	n/a	0.9
F-GFP	pNH051	2.4	ATGAGTAAAGGAGAAGAACTTTTC	[*]TTTGTATAGTTCATCCATGCCATG	pNH040	n/a	0.9
F-YFP	pNH052	2.4	ATGAGTAAAGGAGAAGAACTTTTC	[*]TTTGTATAGTTCATCCATGCCATG	pNH041	n/a	0.9
Mc-mCherry	pNH053	2.4	ATGGTCTCAAAGGGTGAAGAAGAT	[*]GGATCCACTAGTCTTATACAATTC	pNH042	n/a	0.9
mTFP1	pNH054	2.4	ATGGCCGCCTCAAAAGGAGAAGAA	[*]AGCGCTTACGTAGAGCTCGTCCAT	pNH043	pNH013	0.7
N-TAP-tag::[(G₄S)₃]::mTFP1	pNH066	2.6	ATGGTTAAAGAAACAGCAGCAGCG	[*]AGCGCTTACGTAGAGCTCGTCCAT	pNH058	pNH002	0.9
mTFP1::[(G₄S)₃]::C-TAP-tag	pNH094	2.3	ATGGCCGCCTCAAAAGGAGAAGAA	[*]AGCCCATGAGTCCATATGCTGTCT	pNH090	pNH026	0.9
mTFP1::[(G₄S)₃]::2xNLS	pNH070	2.6	ATGGCCGCCTCAAAAGGAGAAGAA	[*]AGCGCTAACTTTTCGCTTCTTCTT	n/a	pNH030	0.8
mTFP1[B]	pNH054	2.4	ATGGCCGCCTCAAAAGGAGAAGAA	[*]AGCGCTTACGTAGAGCTCGTCCAT	n/a	pNH078	0.8
mTFP1[BC]	pNH054	2.4	ATGGCCGCCTCAAAAGGAGAAGAA	[*]AGCGCTTACGTAGAGCTCGTCCAT	n/a	pNH082	0.8

^a sequence to be seamlessly inserted into target; ^b the counter-selection RT-cassette should be PCR-amplified from this construct using, as template, the RT-cassette-containing NotI-NotI fragment; ^c the desired replacement cassette can be excised, as a NotI-NotI fragment, from either the pCC1Fos- (if available) or parental pGOv5-based (Table 1) constructs; ^d size of the isolated NotI-fragment (or NcoI-NcoI fragment for pNH034) to be used as PCR template; ^e suggested 3^′ sequences of ‘recombineering’ ODNs designed to PCR-amplify FP-flanked, RT-cassette-containing sequence. Reverse ODN sequence lacks a 5^′ stop triplet [*]; ^f size of the isolated NotI-fragment to be used to replace the inserted FP-flanked, RT-cassette-containing sequence. All constructs are available from Addgene and are in E. coli EPI300 apart from those built in pGOv5 which are in E. coli DH5α.

ISSN: 1472-6750