Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli

Abstract

We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.

Figure 1

(a) A map of the recombination vector pDF25. A 3.3 kb BamHI fragment of the E.coli recA gene was cloned into the polylinker BglII site of the plasmid pLN135 (9). The plasmid carries an origin of replication (Ori), a temperature-sensitive mutation in the replication initiation protein RepA (RepAts), the gene for resistance to chloramphenicol (Cm^R) and the rpsL+ gene (the wild-type allele of the rpsL gene). Some of the unique cloning sites are indicated. (b) A schematic representation of the sizes of NcoI fragments hybridising to the 2 kb DraI insert following integration of the plasmid pD3γ-lox into PAC 148 insert and its subsequent excision. The original 2.5 kb NcoI fragment is now interrupted by the new NcoI site giving fragments of 1.5 and 1.0 kb. The abbreviations used in the figure are: Bg, BglII; Nc, NcoI; RecA Nc, NcoI site in the recA gene; Cm Nc, NcoI site in the Cm gene; loxP Nc, NcoI site in the loxP oligo; Wt, unmodified PAC; and LoxP, modified PAC with a targeted insertion of the loxP oligo.

Figure 1

(a) A map of the recombination vector pDF25. A 3.3 kb BamHI fragment of the E.coli recA gene was cloned into the polylinker BglII site of the plasmid pLN135 (9). The plasmid carries an origin of replication (Ori), a temperature-sensitive mutation in the replication initiation protein RepA (RepAts), the gene for resistance to chloramphenicol (Cm^R) and the rpsL+ gene (the wild-type allele of the rpsL gene). Some of the unique cloning sites are indicated. (b) A schematic representation of the sizes of NcoI fragments hybridising to the 2 kb DraI insert following integration of the plasmid pD3γ-lox into PAC 148 insert and its subsequent excision. The original 2.5 kb NcoI fragment is now interrupted by the new NcoI site giving fragments of 1.5 and 1.0 kb. The abbreviations used in the figure are: Bg, BglII; Nc, NcoI; RecA Nc, NcoI site in the recA gene; Cm Nc, NcoI site in the Cm gene; loxP Nc, NcoI site in the loxP oligo; Wt, unmodified PAC; and LoxP, modified PAC with a targeted insertion of the loxP oligo.

Figure 2

(a) Southern blot hybridisation of NcoI-digested miniprep DNAs of PAC clones following integration of pD3γlox into PAC 148 insert and probing with the fragment indicated in Figure 1b. Unintegrated pD3γlox DNA digested with NcoI (lane 1), PAC 148 DNA (lane 2) and DNAs of clones selected for the integration of pD3γlox into PAC 148 insert (lanes 3–7). Bands marked with a diamond are diagnostic for the integration. Both the 3 and the 1.2 kb bands are also seen in the unintegrated plasmid (lane 1), consequently they are not marked as diagnostic. The 1.3 kb band is due to cross-hybridisation with the probe. (b) Introduction of the loxP sequence and a diagnostic NcoI site into PAC 148 insert after excision of the plasmid pD3γlox. All DNAs were digested with NcoI and probed as in (a). Unintegrated pD3γlox digested with NcoI (lane 1), PAC 148 (lane 2), DNAs from clones selected following excision of the recombination vector construct pD3γlox (lanes 3–9). The 1.5 and 1.0 kb bands are diagnostic for a successful homologous recombination. The 1.3 kb cross-hybridising band seen in (a) is observed only on longer exposures. (c) Mapping of PAC 148 (lanes 2 and 4) and PAC 148γlox (lanes 1 and 3). DNAs were digested with either NcoI (lanes 1 and 2) or with EcoRI (lanes 3 and 4) and the blot probed with the cosmid clone Cosγγδβ (22). The band marked with an asterisk is a partial digest. The 1.5 and 1.0 kb bands are the result of homologous recombination.

Figure 2

(a) Southern blot hybridisation of NcoI-digested miniprep DNAs of PAC clones following integration of pD3γlox into PAC 148 insert and probing with the fragment indicated in Figure 1b. Unintegrated pD3γlox DNA digested with NcoI (lane 1), PAC 148 DNA (lane 2) and DNAs of clones selected for the integration of pD3γlox into PAC 148 insert (lanes 3–7). Bands marked with a diamond are diagnostic for the integration. Both the 3 and the 1.2 kb bands are also seen in the unintegrated plasmid (lane 1), consequently they are not marked as diagnostic. The 1.3 kb band is due to cross-hybridisation with the probe. (b) Introduction of the loxP sequence and a diagnostic NcoI site into PAC 148 insert after excision of the plasmid pD3γlox. All DNAs were digested with NcoI and probed as in (a). Unintegrated pD3γlox digested with NcoI (lane 1), PAC 148 (lane 2), DNAs from clones selected following excision of the recombination vector construct pD3γlox (lanes 3–9). The 1.5 and 1.0 kb bands are diagnostic for a successful homologous recombination. The 1.3 kb cross-hybridising band seen in (a) is observed only on longer exposures. (c) Mapping of PAC 148 (lanes 2 and 4) and PAC 148γlox (lanes 1 and 3). DNAs were digested with either NcoI (lanes 1 and 2) or with EcoRI (lanes 3 and 4) and the blot probed with the cosmid clone Cosγγδβ (22). The band marked with an asterisk is a partial digest. The 1.5 and 1.0 kb bands are the result of homologous recombination.

Figure 2

(a) Southern blot hybridisation of NcoI-digested miniprep DNAs of PAC clones following integration of pD3γlox into PAC 148 insert and probing with the fragment indicated in Figure 1b. Unintegrated pD3γlox DNA digested with NcoI (lane 1), PAC 148 DNA (lane 2) and DNAs of clones selected for the integration of pD3γlox into PAC 148 insert (lanes 3–7). Bands marked with a diamond are diagnostic for the integration. Both the 3 and the 1.2 kb bands are also seen in the unintegrated plasmid (lane 1), consequently they are not marked as diagnostic. The 1.3 kb band is due to cross-hybridisation with the probe. (b) Introduction of the loxP sequence and a diagnostic NcoI site into PAC 148 insert after excision of the plasmid pD3γlox. All DNAs were digested with NcoI and probed as in (a). Unintegrated pD3γlox digested with NcoI (lane 1), PAC 148 (lane 2), DNAs from clones selected following excision of the recombination vector construct pD3γlox (lanes 3–9). The 1.5 and 1.0 kb bands are diagnostic for a successful homologous recombination. The 1.3 kb cross-hybridising band seen in (a) is observed only on longer exposures. (c) Mapping of PAC 148 (lanes 2 and 4) and PAC 148γlox (lanes 1 and 3). DNAs were digested with either NcoI (lanes 1 and 2) or with EcoRI (lanes 3 and 4) and the blot probed with the cosmid clone Cosγγδβ (22). The band marked with an asterisk is a partial digest. The 1.5 and 1.0 kb bands are the result of homologous recombination.

Figure 3

PAC 100 (lane 1) and PAC 148 (lane 7) grown in the absence of RecA and the recombinases RecE and RecT. The effect of either RecA (lanes 2, 5, 8 and 11) or the recombinases RecE and RecT (lanes 3, 6, 9 and 12) on PAC 100 and 148 inserts. DNAs were digested with either XhoI (lanes 1–3 and 7–9) or ClaI (lanes 4–6 and 10–12), run on a 0.4% agarose gel in TBE buffer (8) and visualised by staining with 0.5 µg/ml ethidium bromide.