The Journal of Neuroscience, April 6, 2005, ():
Plexin-A4 Mediates Axon-Repulsive Activities of Both Secreted and Transmembrane Semaphorins and Plays Roles in Nerve Fiber Guidance
J. Neurosci. Suto et al.
25: 3628
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Supplemental Figure 1. Generation of plexin-A4 mutant mice by targeted disruption of the plexin-A4 gene.
(A) Isolation of the plexin-A4 genomic DNA. We screened a genomic DNA library with two PCR fragments of plexin-A4 cDNA (corresponding to 825th-1150th bases and 1826th-2142nd bases of the plexin-A4 cDNA), and isolated a DNA fragment that contains an exon (Ex) encoding the signal peptide (S) and most of the sema domain (SD) of the plexin-A4 protein (corresponding to 887th-2158th bases of the plexin-A4 cDNA). C1, C2 and C3, 3 cysteine clusters/Met-related sequences (MRSs) in the extracellular segment of the protein; TM, transmembrane domain; CD, cytoplasmic domain.
(B) Plexin-A4 gene-targeting strategy and production of plexin-A4 mutant mice. The wild-type plexin-A4 allele (WT), the targeting vector and the targeted locus are shown. In the targeting vector, most of the exon [the region between the restriction sites for Pvu I (Pv) and Pst I (Ps) shown in the wild-type allele] including the coding region for the sema domain was deleted, and instead the loxP-IRES Tak PGK-neo cassette (ITak-neo) was inserted for positive selection. The diphtheria toxin A fragment gene cassette (DT) was inserted upstream of the plexin-A4 genome for negative selection. ?, the position of the initiation codon, ATG; Nh and H, the restriction sites for Nhe I and Hind III, respectively; BSK, pBluescript. The targeting vector (20 µg) linearized by Not I was electroporated into 1x107 TT-2 cells. Homologous recombinants were selected using G418 (200 µg/ml). Successful targeting was confirmed by Southern blotting using a 5’ flanking probe shown in B. Targeted TT-2 ES cells were microinjected into 8-cell stage ICR mouse embryos. The embryos were cultured in M16 medium overnight until the blastocyst stage, and then were transplanted into recipient ICR mice. Plexin-A4 heterozygous (PlexA4+/-) mice were obtained by crossing germline chimeric mice with mice of the C57BL/6 strain, and backcrossed eight to ten times. Plexin-A4 homozygous (PlexA4-/-) mice were obtained by intercrossing the heterozygous offspring.
(C) Genotyping. Genotypes of mice were determined by PCR using primers P1 (5’-GTAGAGGTGCCCATTGGCTGTGAGCGC-3’), P2 (5’-GCCAACAAGAGACCAGAATGAGGTGCC-3’) corresponding to the plexin-A4 genome sequences, and P3 (5’-TACCCGTGATATTGCTGAAGAGCTTGG-3’) corresponding to the neomycin resistance genes (see B). DNAs of expected size (511 bps for the wild-type allele, and 857 bps for the mutant allele) are amplified.
(D) Southern blot analysis of embryos derived from plexin-A4 heterozygous intercrosses. The genomic DNAs were digested with Nhe I and probed with the 5’ probe shown in B. Expected sizes of DNAs of the wild-type and mutant alleles (6 kbp and 4 kbp, respectively; see B) are indicated.
(E) Production of anti-plexin-A4 antibodies and immunoblot analysis. Antibodies were produced against the fusion proteins for the IPT1 domain of mouse plexin-A4 (amino acids 852-948; see A). The sequences that encode the IPT1 domain were inserted into the expression vector pET-23a+ (Novagen, Darmstadt, Germany). The six histidine-tagged fusion proteins were produced in the BL21-CodonPLUS(DE3)-PIL strain of E. Coli (Stratagene, La Jolla, CA), and purified using Ni-NTA agarose (QIAGEN, Hilden, Germany). Rabbits were immunized with the fusion proteins. IgG was purified using HiTrap Protein A HP (Amersham Biosciences). The serum antibodies recognize a band at around 200 kd (indicated by an arrow) in HEK293T cells that were transfected with plexin-A4 cDNA (A4) but not cells with plexin-A1 (A1), plexin-A2 (A2) or plexin-A3 (A3) cDNAs or the vector only (v). The antibodies also recognize a band at around 200 kd in membrane fractions of brains from E15 wild-type (+/+) and plexin-A4 heterozygous (+/-) but not plexin-A4 homozygous (-/-) embryos. These results suggest that the present plexin-A4 gene-knockout mouse is a null mutant.
