Journal: The Journal of Neuroscience
Article Title: Mice Deficient in Collapsin Response Mediator Protein-1 Exhibit Impaired Long-Term Potentiation and Impaired Spatial Learning and Memory
Figure Lengend Snippet: Generation and characterization of CRMP-1−/− mice. A, The genomic structure of CRMP-1 and the targeted alleles. The CRMP-1 exons 2–6 (filled boxes) are depicted along with restriction enzyme sites (A, AflII; Ap, ApaI; B, BamHI; C, ClaI; H, HindIII; N, NcoI; X, XhoI; R, EcoRI). The wild-type allele (+) targeted by vector I generated the allele (“t”) harboring a loxP (filled triangle)-flanked HPRT (hatched box) in intron 5. Cre excision generated the one-loxP allele. Subsequent transfection of the one-loxP-containing ES cells with vector II generated the floxed allele (fl). Subsequent excision by Cre deleted the HPRT and exons 4 and 5 [the null allele (−)]. Open bars, Probes A and B for Southern blotting. Primers (2U, 3U, 3D, 4U, 4D, 5D, and 6D) for genotyping by PCR. C* in vector I, Deleted ClaI site. B, Southern blotting of recombinant ES cell clones digested by AflII and AflII plus BamHI and detected by probes A and B. DNA in each lane, 5–10 μg. Only probe A results are shown. Lane 1, Wild-type ES cells (showing 8.3 kb “+” allele). Wild-type allele is omitted in the following descriptions. Lane 2, Vector I targeted clone (10.8 kb; “t” allele). Lanes 3–5, DNA from ES cells of lane 2 after cre-excision and successful vector II recombination (lane 3, 9.8 kb floxed “fl” allele), unsuccessful vector II recombination in cis (lane 4, 6.8 kb “loxP” allele), and successful vector II recombination in trans (lane 5, a loxP allele plus a t* allele denoting the clone with the loxP-flanked HPRT fragment in intron 3 of the wild-type allele of lane 4). Lane 6, ES cell clone in lane 7 after Cre transfection to generate the CRMP-1−/− allele, the (−) allele. Lane 7, ES cell clone in lane 3 by AflII. C, Southern blotting of AflII-digested tail DNA from pups derived from ES cell clones of lane 6 of panel B. +/+, +/−, and −/−, Wild-type, heterozygous, and homozygous CRMP-1−/− mice. Both probes A and B gave the same pattern. D, Northern blotting of 20 μg of total RNA from P7 mouse brains and kidneys, using exons 1–11 CRMP-1 cDNA and a GAPDH probe. M, RNA size maker (MBI Fermentas, Hanover, MD). E, RT-PCR analysis. Total RNA (P7) of brains and PCR primers 2U and 6D were used. The 582 and 356 bp bands represent the wild-type and knock-out alleles, respectively. F, Western blot analysis of CRMPs in whole-brain extracts from CRMP-1+/+ and CRMP-1−/− mice at P1. Tissue extract samples (15 μg) were loaded in each lane. The membrane was first reacted with anti-CRMP-1 antibody (in-house), followed by anti-CRMP-2, anti-CRMP-4, and then anti-actin antibodies (internal control) with stripping and washing steps in between each antibody reaction. Rat P1 brain, Positive control. Lanes +/+, +/−, and −/−, Wild-type, heterozygous, and homozygous CRMP-1 knock-out mice, respectively. Multiple CRMP bands were attributable to phosphorylation events.
Article Snippet: The antibodies used were anti-CRMP-1 (prepared in-house or purchased from Millipore), anti-CRMP-2 (prepared in-house), anti-CRMP-4 (BD PharMingen, San Diego, CA), anti-growth-associated protein-43 (GAP-43) (Abcam, Cambridge, UK), and anti-microtubule-associated protein 2 (MAP2) and postsynaptic density-95 (PSD95) (Millipore) and anti-actin polyclonal antibodies (Sigma, St. Louis, MO).
Techniques: Plasmid Preparation, Generated, Transfection, Southern Blot, Recombinant, Clone Assay, Derivative Assay, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, Stripping Membranes, Positive Control