R26creer mice

Prdm1^f/f mice (11 (link)) were crossed with CD11c-Cre or R26CreER mice, both purchased from The Jackson Laboratory, to generate Prdm1^f/fCD11c-Cre^+/− (CKO-11c), Prdm1^f/fER-Cre^+/− (CKO-ER), and their littermate control Prdm1^f/fCD11c-Cre^−/− (Ctrl-11c) or Prdm1^f/fER-Cre^−/− (Ctrl-ER) mice. To avoid the autoimmune phenotypes of female CKO-11c mice (9 (link)), only male CKO-11c and male littermate control mice were used in all experiments. Tlr7 knockout (KO) (12 (link)) and Blimp-1-yellow fluorescent protein (YFP) reporter mice (13 (link)) were purchased from The Jackson Laboratory, and Tlr9 KO (obtained from Dr. Shizuo Akira) (14 (link)) mice were paired with wild-type C57BL/6 mice (purchased from the National Laboratory Animal Center, Taipei, Taiwan). All mice were housed and bred in the specific pathogen free conditions in the animal facility of Institute of Cellular and Organismic biology at Academia Sinica. Animal experimental protocols were approved by IACUC of Academia Sinica.

All animal procedures (animal handling, experimental procedure, anesthesia and euthanasia) were conducted in accordance with the University of Nebraska-Lincoln’s Institutional Animal Care and Use Committee and approved by the Institutional Animal Care Program (protocol 1229). Studies were conducted in female Balb/c mice (Jackson Laboratory, stock number 000651), ages 8 to 20 weeks, unless noted otherwise. Exosome and Cargo Tracking (ECT) mice were developed in our laboratory and express an exosome marker protein (CD63) fused to eGFP. The presence of the CD63/eGFP gene was confirmed by PCR (Table 1); expression of the transgene was confirmed by imaging green fluorescence (excitation 455–495 nm, emission 503–523 nm). Homozygous Drosha knockout mice^{52 (link)}, a gift by Dr. Tatsuya Kobayashi (Massachusetts General Hospital), were mated to tamoxifen-inducible R26CreER mice (Jackson Labs, stock number: 004847). Homozygous tamoxifen-inducible Drosha knockout mice were identified by PCR (Table 1). Transgenic pigs with ubiquitous ZsGreen1 expression were developed as described previously^{53 (link)}.Table 1

PCR primers.

Primers	Gene
5′-GCAGAAAGTCTCCCACTCCTAACCTTC-3′ (F)	Drosha
5′-CCAGGGGAAATTAAACGAGACTCC-3′ (R)
5′-AAGGGAGCTGCAGTGGAGTA-3′ (F)	Cre
5′-CCGAAAATCTGTGGGAAGTC-3′ (R)
5′-TCTTGCGAACCTCATCACTC-3′ (R)
5′-GCAAGAGGTGCGGAAGATTA-3′ (F)	CD63/eGFP
5′-GGATGGCGAAGCTAAGATCAA-3′ (R)

Animal studies were conducted in AAALAC-approved facilities according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Washington State University. C57BL/6 mice, PDGFRαCreER (stock number: 018280), VEGFR2^loxP (stock number: 018977), and R26CreER mice (stock number 004847) and ROSA^mT/mG mice (stock number: 007676) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). R26-RARα403 mice were kindly provided by Dr Cathy Mendelsohn [42 (link)]. All mice were fed with a diet containing 15 IU g⁻¹ Vitamin A (Teklad global diet 2018).
Retinoic acid (10 mg kg⁻¹ BW), BMS493 (10 mg kg⁻¹ BW) or vehicle (DMSO) dissolved in corn oil were intraperitoneally injected once per two days for mouse experiments. VEGF164 was dissolved in phosphate-buffered saline (PBS) at a dose of 2 μg kg⁻¹ BW per injection [43 (link)]. PDGFRα tracking mice were given a single intraperitoneal injection of 100 mg kg⁻¹ BW tamoxifen to label PDGFRα⁺ cells. Conditional knockouts were induced by daily intraperitoneal injection of 25 mg kg⁻¹ BW tamoxifen for three days. Mice lacking Cre or lox genes were used as the WT control for transgenic mice; both control and Cre-lox mice were injected with tamoxifen. The doses of chemicals were determined by our preliminary experiments.