pbr322 backbone  (Addgene inc)

Journal: PLoS ONE

Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations

doi: 10.1371/journal.pone.0007757

Figure Lengend Snippet: (A) The 5′ end of the previously produced unconditional Ate1 − allele , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).

Article Snippet: The entire ∼12 kb HindIII fragment and a part of the ∼2.9 kb fragment were modified as described below and assembled into a final ∼22.5 kb targeting vector consisting of the following parts ( ): ( i ) pBR322 backbone (New England Biolabs, Ipswich, MA); ( ii ) a ∼6.3 kb “long arm” of Ate1 homology containing the Ate1 exon 1a, the bidirectional P Ate1 promoter , and exon 1b; ( iii ) A single loxP site ∼300 bp upstream of Ate1 exon 2; ( iv ) a ∼2 kb fragment that contains, 50 bp downstream of Ate1 exon 4, a “floxed” Hph (hygromycin) antibiotic-resistance marker, expressed from the P PGK promoter ; ( v ) a ∼1.2 kb “short arm” of Ate1 homology that spans most of the intron between exons 4 and 5; ( vi ) a gene encoding HSV-TK (herpes simplex virus thymidine kinase), expressed from the P PGK promoter.

Techniques: Produced, Selection, Marker, Construct, Sequencing, Plasmid Preparation, In Vivo, Expressing, Hybridization