empty vector control virus  (Addgene inc)

Journal: Genes & Diseases

Article Title: E674Q (Shanghai APP mutant), a novel amyloid precursor protein mutation, in familial late-onset Alzheimer's disease

doi: 10.1016/j.gendis.2023.02.051

Figure Lengend Snippet: The E674Q mutation facilitates the fragmentation of APP by BACE1 in vitro and in vivo . (A) Western blot analysis of HEK293 cells transfected with wild-type (WT) APP, APP E674Q, and APP with the Swedish mutation. (B, C) The levels of C99 and C83 fragments were increased in cells transfected with APP E674Q and APPswe. (D, H) ELISA quantification of the concentrations of Aβ40 (D) and Aβ42 (H) in conditioned medium from HEK293 cells transfected with wild-type and mutant APP. Data are presented as mean ± S.E.M. P < 0.01, one-way ANOVA. (E) Western blot analysis of AAV-mediated injection mice with APPwt, APPE674Q, and APPswe (Y188, ab32136, Abcam). (F, G) The levels of APP and C99 fragment were increased in mice hippocampus injected with AAV-APPE674Q and AAV-APPswe. (I–O) Behavioral experiments 6 months after the mice were injected. In the novel object recognition test, DR2 of APPE674Q animals was less than the control ( P < 0.05; J, K); in the Y maze test, APPswe ( P < 0.05) and APPE674Q ( P < 0.01) had a significant difference from the control but no noticeable difference from APPwt (L); in the Barnes maze test, the two APP mutation groups show a potentially faster learning trend than APPwt group (M, N); in the testing trail, APPswe ( P < 0.01) group had a noticeable shorter latency to escape than APPwt group (O).

Article Snippet: Adeno-associated virus vectors (AAVrh.9) encoding human mutant full-length APPswe, APPE674Q, APPwt, and empty control were purchased from Sangon Biotech, China.

Techniques: Mutagenesis, In Vitro, In Vivo, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Injection, Control

Journal: Journal of Clinical Investigation

Article Title: H3K27me3 dynamics dictate evolving uterine states in pregnancy and parturition

doi: 10.1172/jci95937

Figure Lengend Snippet: Figure 2. H3K27me3 accrual in DSCs attenuates type 1 immunity in the decidua. (A) H3K27me3 tracks at the Cxcl9, Cxcl10, Cxcl12, Cxcl16, and Csf1 loci. The tracks are the pileups of the 3 independent replicates. The log2 concentration for each called peak and the TSS with direction of transcription (arrow) are indicated. Sequencing reads from total input chromatin were homogeneous across these loci (not shown). (B–J) H3K27me3 silences Csf1 in DSCs, limiting macrophage accumulation in the decidua. (B–D) Immunostaining for F4/80+ macrophages (red) in the undecidualized E3.5 uterus 1 day prior to implantation (B), an interimplantation site on E6.5 (C), and an E6.5 implantation site (D). Note the dramatically lower tissue density of macrophages within the decidua as compared with the undecidualized endometrium, consistent with previous results at later gestation (9). Representative images from 3 mice/group; panels C and D show sections near those in Figure 1, H and I, respectively. (E) qRT-PCR determination of Csf1 mRNA expression in stromal cells isolated from E7.5 pregnant mice and cultured for 24 hours (mean ± SEM; n = 3 samples/group). DSCs express lower levels of Csf1 than MSCs, at least in part explaining the lower level of Csf1 expression in the whole decidua (9). (F–J) Effect of ectopic CSF-1 expression within the decidua. Artificially decidu- alized uteri were injected with Csf1-expressing or empty vector control lentivirus on the day corresponding to E5.5 and the mice were sacrificed 2 days later. Viral preparations included aliquots of EGFP reporter lentiviruses to identify transduced areas. Representative images of serial sections immunostained for F4/80 or GFP (F–I) and mean ± SEM of quantified F4/80+ cell densities in infected (GFP+) and uninfected (GFP–) decidual areas (J) from 4 control virus– infected mice and 5 Csf1 virus–infected mice. Note that decidual areas infected with Csf1-expressing lentiviruses accumulate macrophages, demonstrating a CSF-1 deficit within this tissue layer. Asterisk indicates decidual lumen.

Article Snippet: 75 × 107 Csf1-expressing or empty vector control virus mixed with 30 × 107 lentiviral particles produced from the FG12 EGFP reporter construct (Addgene, no. 14884), was injected into artificially decidualized uteri of pseudopregnant mice at multiple locations on E5.5 via a transmural approach, using our previously described methods (7).

Techniques: Concentration Assay, Sequencing, Immunostaining, Quantitative RT-PCR, Expressing, Isolation, Cell Culture, Injection, Plasmid Preparation, Control, Infection, Virus