C57BL6 mice were purchased from Clea Japan (Tokyo, Japan) or SLC (Shizuoka, Japan). FX Friuli−/− mice (Tai et al, 2008 ) and KikGR mice (Tomura et al, 2014 ) were used in experiments. The genetic background of KikGR mouse is C57BL/6. FX Friuli−/− mice were initially introduced as 129Sv/C57BL6/J and backcrossing the offspring with C57BL6/J mice at least three times. To minimize the effect of genetic background, we used littermates in the experiments of FX Friuli−/− mice. Green fluorescent protein (GFP)‐Tg (C57BL/6‐Tg (CAG‐EGFP)C14‐Y01‐FM131Osb) mice were obtained from the RIKEN Bio Resource Center. Mice were housed in a specific pathogen‐free condition at a constant temperature and humidity with a 12 h‐12 h light–dark cycle. All animal procedures were performed according to the guidelines of the Animal Research Committee of Tokyo Women's Medical University. For animal studies, the investigators were not blinded allocation during experiments and outcome assessment.
C57bl 6 tg cag egfp c14 y01 fm131osb
Manufactured by RIKEN BioResource Center
Sourced in Japan
C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb is a transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) under the control of the cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG) promoter. This strain can be used for various biological applications where visualization of cells or tissues is required.
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4 protocols using c57bl 6 tg cag egfp c14 y01 fm131osb
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Murine Genotypes for Biological Research
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Isolation of Adipose-Derived Stem Cells
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All procedures were approved by the Research Ethics Committee of The University of Tokyo Hospital (ethical permission number 622). ASCs were isolated from 6- or 7-week-old EGFP transgenic mice (C57BL/6-Tg(CAG-EGFP)C14–Y01-FM131Osb; RIKEN BioResource Center, Japan). Adipose tissue was harvested from the inguinal fat pads, minced, and digested by shaking with 0.1% collagenase solution for 30 min at 37 °C. The solution was filtered through a cell strainer (70-μm pore size; BD Falcon, Japan) and centrifuged at 250G for 5 min, and the supernatant was removed to obtain the stromal vascular fraction. Cells were seeded at 1.0 × 106 cells/dish in 100-mm dishes, and cultured in medium consisting of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Sigma–Aldrich,USA), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (Sigma–Aldrich) at 37 °C in 5% CO2. The medium was changed every 3–4 days and cells were passaged using trypsin–EDTA (Sigma–Aldrich), when they reached 80–90% confluence. After 2 passages, ASCs were used for transplantation.
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Obtaining GFP-expressing and Wild-type Fetuses for Microchimerism Studies
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The inbred strains, BALB/cByJJcl and C57BL/6JJcl were obtained from Clea Japan. GFP expressing mice, C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb was obtained from RIKEN BioResource Research Center (RBRC), which was developed by Okabe M. et al. [19 (link)] and genotyping was performed according to the instructions provided by RBRC using PCR. To obtain GFP heterozygous female mice, BALB/c female mice were mated with GFP homozygous male mice. To obtain wild type fetus with MMc cells, GFP heterozygous female mice were mated with BALB/c male mice and only fetuses without GFP genes were used for experiments (Fig 1A and 1B ). Fetuses in the pregnant C57BL/6JJcl female mice were used as negative control for the MMc cell counting experiment.
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Maternal Microchimerism Detection Protocol
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BALB/cByJJcl inbred strain was obtained from Clea Japan. GFP-expressing mice, C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb, developed by Okabe et al.21 (link), were from RIKEN BioResource Research Center (RBRC). Genotyping was performed according to the instructions provided by RBRC using polymerase chain reaction (PCR). To obtain wild-type fetuses with GFP-positive MMc cells, GFP-heterozygous female mother mice were mated with BALB/cByJJcl, GFP−/− male mice, and only fetuses lacking the GFP gene (n = 52) were used in subsequent experiments (Fig. 1 ) in detecting GFP+/− maternal cells. Furthermore, to prevent detection of GFP-positive grand-maternal cells39 , the mother mice were obtained by crossing BALB/cByJJcl wild-type female mice with GFP-homozygous male mice. MHC types of mice were carefully designed for utility to concentrate MMc cells using magnetic cell sorting system (Fig. 2 ).
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