Figure 1.
Details of the genetic tools used.
(A) Structure and characteristic elements of the multicopy cloning vector pHTH22 with unique restriction sites indicated. The gene region encoding HSV thymidine kinase (TK) under the control of mouse phosphoglycerate kinase promoter and terminator is shown with a black curved arrow. The vector portion of the plasmid including the gene for ampicillin resistance (AmpR) and pUC19 origin of replication (ori) are shown with gray symbols (curved arrow and rectangle). (B) The nucleotide sequence of the polylinker and its flanking regions in pHTH22. The polylinker is shown in boldface with enzyme recognition sites underlined. The arrows indicate the binding sites of primers that were used for the confirmatory sequencing of genomic inserts' ends. (C) Structures of the Kan/Neo-loxP-Mu and Kan/Neo-loxP-FRT-Mu transposons. The transposons contain bacterial (pbact) and eukaryotic (pSV40) promoters (short arrows), a marker gene (Kan/Neo) conferring resistance to kanamycin in bacteria and G418 in eukaryotes (black arrow), and the HSV thymidine kinase polyadenylation (HSV TK polyA) signal (small rectangle). The loxP sites are indicated by triangles and FRT sites by pentagons. The rectangles in the transposon ends indicate 50 bp of Mu R-end DNA sequences in inverted orientation relative to each other. For the sake of clarity, the features are not in scale. The BglII sites in the ends are used to excise the transposons from their carrier plasmids. The sequences of the transposon-containing plasmids pHTH19 (Kan/Neo-loxP-Mu) and pHTH24 (Kan/Neo-loxP-FRT-Mu) are available upon request.
Figure 2.
Flowchart for the construction of conditional knockout vectors.
Part A. (i) an entire BAC clone is initially digested with an appropriate restriction endonuclease, and (ii) the ensuing fragment pool is then used as a target for the first transposition reaction. (iii) The fragment pool is subsequently ligated into a suitable vector plasmid, and those clones that include transposon-containing BAC fragments are selected using both the transposon marker and vector marker. Next, (iv) a plasmid clone that contains a transposon insertion in a desired location is identified by a PCR screen with appropriate primers, one transposon specific and the other target specific. (v) The chosen plasmid is then introduced into an E. coli strain that expresses Cre recombinase. The selectable marker is eliminated by Cre recombination in vivo, leaving a single loxP site in the construction. Part B. The second transposition reaction (vi) using pre-assembled transposition complexes (vii) introduces into the construction a marker gene that is flanked by loxP and FRT sites. (viii) As above, a suitable clone is screened by PCR to identify a plasmid, which contains the second transposon inserted in a suitable location and orientation on the opposite side (to the first transposon) of the exon of interest. Genomic DNA is shown with a black line, and the orange rectangle denotes an exon. The transposons are shown with black bars featuring the Kan/Neo cassette (green arrow), loxP sites (white triangles), FRT sites (white pentagons), and transposon ends (black rectangles).
Figure 3.
PCR screening of transposon-containing plasmid clones.
(A) Cdh22 locus. The first transposon insertion was screened using the primer pair 1B5′/1B3′. The second transposon insertion was screened using the primer pair 2B5′/HSP430. (B) Drapc1 locus. The first transposon insertion was screened using the primer pair N2/HSP507. The second transposon insertion was screened using the primer pair HSP430/HSP518. In both panels, the relevant distances from the identified transposon insertion site to the 5′ ends of the primers are shown in base pairs (bp). For the sake of clarity, figure elements are not drawn in scale.
Figure 4.
Verification of the functionality of the Cre/loxP and Flp/FRT site-specific recombination systems in the context of the gene targeting constructions.
The final constructions were introduced into E. coli strains 294-Cre and 294-Flp, expressing Cre and FLP recombinase, respectively [28]. From each strain, two independent plasmid isolates, marked C1 and C2 (from the 294-Cre strain) or F1 and F2 (from the 294-Flp strain), were subjected to restriction analysis (on the left). On these analyses, the original targeting plasmid is marked with T. On the right are shown the respective plasmid maps with the relevant restriction enzyme as well as LoxP and FRT sites indicated (BamHI, XbaI and HindIII sites are indicated with B, X and H, respectively). (A) Analysis of the targeting construction for the Cdh22 locus. As the size marker serves the XbaI digestion of the respective targeting construction. (B) Analysis of the targeting construction for the Drapc1 locus. As the size marker serves the HindIII digestion of the respective targeting construction.
Figure 5.
Targeting strategy for the conditional inactivation of the Cdh22 gene.
(A) Restriction maps of the mouse wild type Cdh22 locus, the targeting vector, and the targeted allele. Relevant restriction sites: E, EcoRI; K, KpnI. Vertical dotted lines highlight homologous regions. (B) Southern analysis of genomic DNA from ES cells (EcoRI digestion), with the 5′-probe illustrating the 21.5 kb wild type (wt) and 10.5 kb targeted (cond) alleles. (C) Southern analysis of genomic DNA from ES cells (EcoRI digestion), with the 3′ probe illustrating the 21.5 kb wild type (wt) and 8.9 kb targeted (cond) alleles.
Figure 6.
Targeting strategy for the conditional inactivation of the Drapc1 gene.
(A) Restriction map of the mouse Drapc1 locus, the targeting vector and the targeted allele. Relevant restriction sites: A, AvrII; B, BamHI; N, NsiI. Vertical dotted lines highlight homologous regions. (B) Southern analysis of genomic DNA from ES cells (BamHI digestion), with the 5′ probe illustrating the 6.4 kb wild-type (wt) and 4.9 kb targeted (cond) alleles. (C) Southern analysis of genomic DNA from ES cells (AvrII digestion), with the 3′ probe illustrating the 14.2 kb wild type (wt) and 6.4 kb targeted (cond) alleles.
Figure 7.
Comparison of wild type, conditional, and deletion alleles of the mouse Cdh22 locus.
(A) A schematic of wild-type (wt), conditional (cond), and Cre-recombined (del) Cdh22 alleles. (B) Southern blot analysis of DNA isolated from the tails of mice carrying different Cdh22 alleles (EcoRV digestion), with the 3′probe illustrating the 14.6 kb wild type (wt), 17.0 kb conditional (cond), and 12.6 kb deletion (del) alleles.
Figure 8.
Comparison of wild type, conditional, and deletion alleles of the mouse Drapc1 locus.
(A) A schematic of wild-type (wt), conditional (cond), and Cre-recombined (del) Drapc1 alleles. (B) Southern blot analysis of DNA isolated from the tails of mice carrying different Drapc1 alleles (AvrII digestion), with the 3′probe illustrating the 14.2 kb wild type (wt), 6.4 kb conditional (cond), and 8.3 kb deletion (del) alleles.
Table 1.
Oligonucleotides used in this study.