restriction cloning with pmk411 ostir1 f74g maid egfp nluc cassette  (Addgene inc)

Journal: Development, Growth & Differentiation

Article Title: Establishment and characterization of mouse lines useful for endogenous protein degradation via an improved auxin‐inducible degron system ( AID2 )

doi: 10.1111/dgd.12942

Figure Lengend Snippet: Reversible protein knockdown in adult organs and developing embryos. (a, b) Western blot analyses showed reduction and recovery of AID‐EGFP in the heart (a) and brain (b) upon administration of 5‐Ph‐IAA (5 mg/kg). Samples were prepared 1, 5, 24, 48, and 72 h after injection into adult pMK411#19 mice. An E9.5 embryo sample of pMK411#19 was used as a loading control. The same amount of protein (30 μg) was loaded. Each picture was taken before sample preparation. Scale bar, 1 mm. (c) Comparison of two transgenic (Tg) lines, pMK411#16 and #19. Embryos recovered from pregnant mothers crossed with each male Tg with (+) and without (−) injection of 5‐Ph‐IAA (5 mg/kg). EGFP reporter intensities were measured 1 h after injection and the intensities were compared. Scale bar, 1 mm. (d) Protein knockdown and recovery in developing embryos visualized by EGFP expression. 5‐Ph‐IAA (1 mg/kg) was injected into pregnant MCH mothers crossed with pMK411#16 at E10.5 (for 72 h), E11.5 (for 48 h), E12.5 (for 24 h), and E13.5 (for 2 h), and all embryos were examined at E13.5. Scale bar, 1 mm. Quantification data are shown as a bar graph. Mean intensities were calculated by subtracting the values of control (Tg‐negative) embryos in each litter.

Article Snippet: One microgram of genomic DNA prepared from pMK411#16 and #19 was digested with Hae III and self‐ligated with T4 ligase (TAKARA, 2011A).

Techniques: Knockdown, Western Blot, Injection, Control, Sample Prep, Comparison, Transgenic Assay, Expressing

Journal: Development, Growth & Differentiation

Article Title: Establishment and characterization of mouse lines useful for endogenous protein degradation via an improved auxin‐inducible degron system ( AID2 )

doi: 10.1111/dgd.12942

Figure Lengend Snippet: Characterization of a TIR1‐reporter line, TG‐CAG‐AID‐mCherry. (a) A male TG‐CAG‐AID‐mCherry mouse was crossed with pMK411#16 or #19 females. (b) Fluorescence signals of EGFP and mCherry were examined in E13.5 embryos from pregnant mothers with or without administration of 5‐Ph‐IAA (5 mg/kg) for 5 or 20 h. The genotype of each embryo (shown as + or –) was determined by polymerase chain reaction (PCR) after imaging. Scale bar, 1 mm. Quantitative data of fluorescent signals are shown below pictures. mCherry intensities were calculated using a similar method to Figure . (c) Western blotting analyses of transgene expression. TIR1(F74G) was detected by an anti‐TIR1 antibody. AID‐EGFP was detected by an anti‐AID antibody. AID‐mCherry signal was analyzed by anti‐AID and anti‐mCherry antibodies. β‐actin was an internal control. The expected protein size is indicated on the left side of the gel. pMK411#19 without crossing with TG‐CAG‐AID‐mCherry was used as a control.

Article Snippet: One microgram of genomic DNA prepared from pMK411#16 and #19 was digested with Hae III and self‐ligated with T4 ligase (TAKARA, 2011A).

Techniques: Fluorescence, Polymerase Chain Reaction, Imaging, Western Blot, Expressing, Control

Journal: Development, Growth & Differentiation

Article Title: Establishment and characterization of mouse lines useful for endogenous protein degradation via an improved auxin‐inducible degron system ( AID2 )

doi: 10.1111/dgd.12942

Figure Lengend Snippet: Endogenous AID‐tagged protein can be degraded quickly in vivo. (a) The homozygous AID‐DCP2 female mouse was crossed with an AID‐DCP2/pMK411‐#19 double heterozygous male. (b) Western blot analyses of E15.5 brain lysate for the detection of AID‐tagged DCP2 by anti‐AID and anti‐DCP2 antibodies. Pregnant female mice received intraperitoneal administration of 5‐Ph‐IAA (5 mg/kg) at E14.5 (for 20‐h treatment) and E15.5 (for 5‐h treatment). Genotype was indicated on each gel line. A/A indicates homozygous AID‐DCP2, A/+ indicates heterozygous AID‐DCP2. TIR1 indicates the presence of pMK411‐#19 allele. (c) Immunofluorescence images of embryonic brains treated with 5‐Ph‐IAA for 20 h (same sample used as for (b)). P‐body signals were detected by anti‐DDX6 (green). A/A indicates homozygous AID‐DCP2, and A/A(+TIR1) indicates homozygous AID‐DCP2 including pMK411#19. Nuclear staining was conducted using Hoechst 33342 (blue). The scale bar indicates 20 μm.

Article Snippet: One microgram of genomic DNA prepared from pMK411#16 and #19 was digested with Hae III and self‐ligated with T4 ligase (TAKARA, 2011A).

Techniques: In Vivo, Western Blot, Immunofluorescence, Staining