Pdx1-GFP mouse embryo: Visualization of GFP fl uorescence through the intact e10.5 Pdx1(GFP/w) embryo, showing phase ( a ), GFP ( b ), and merged images ( c ) of a representative embryo. The arrowhead ( c ) indicates expression of the GFP reporter in the developing pancreas. The developing pancreatic buds are shown in ( d ) and ( e ). Abbreviations: dp dorsal pancreas, vp ventral pancreas, g gut-tube/duodenum 

Context in source publication

Context 1

... 6-Well Low Adherence Plates (Corning). Media conditions described are used to facilitate differentiation of progenitors into islet-like cell aggregates (ICAs) using a method previously described [ 12 – 16 ] with few modifi cations ( see Note 4 ). 1. Serum-free D0 (2) media: DMEM/F12 media without phenol red (Gibco) ( see Note 5 ), 1 % (w/v) BSA (Sigma), 1× ITS (Gibco) + additives ( see Table 1 ). 2. Serum-free D7 (2) media: DMEM/F12 media without phenol red (Gibco), 1 % (w/v) BSA (Sigma), 1× ITS (Gibco), 6.0 mM taurine (Sigma), 200 nM GLP-1 agonist (exendin-4; Sigma), 2 mM nicotinamide (Sigma). 3. Black opaque 96-well fl at-bottom microplates for lumines- cence and fl uorescence assays (Corning). Embryo dissection protocols described in this manuscript are adapted from those described by Shea et al. [ 17 ]. Several protocols described in detail herein are adapted, modifi ed, and optimized from analogous protocols previously published from our laboratory [ 18 ]. 1. Pdx1(GFP/w) mice are housed within a Physical Containment Level 2 (PC2) facility, using IVC caging systems. Successful embryos are generated by mating healthy wild-type (w/w) female mice with heterozygous [Pdx1(GFP/w)] mice at an optimum breeding age of 8–12 weeks ( see Note 6 ). 2. Elements of the physical and social environment of the animals are vital factors in optimizing a successful breeding outcome in transgenic mouse models. Animals are housed together for company where possible. Enrichment is provided in the form of tissue papers and cardboard boxes placed inside the cages to provide important stimuli to encourage natural breeding behaviour and to provide a secure feeling environment for the pregnant female mice. Animals are monitored daily, but handling and noise disturbance is kept to a minimum to reduce any unnecessary distress to the animals that may adversely affect the breeding outcomes. 3. The transgenic mouse model for Pdx1-GFP is available to monitor the expression of the pancreas and duodenal homeobox gene 1 in vivo and during differentiation of cells into an endocrine pancreatic lineage. Pdx1(GFP/w) mice are kindly provided by Prof. Edouard Stanley and Prof. Andrew Elefanty (Murdoch Children’s Research Institute and Monash University, Melbourne) and are maintained by the group of A/Prof. Anand Hardikar. Progenitor populations isolated from these Pdx1 (GFP/w) mice express GFP under the control of Pdx1 promoter as they commit to a pancreatic lineage. They also continue to express GFP as cells begin to transcribe insulin, since Pdx1 is also transcribed by mature insulin- producing cells. For further details regarding the transgenic animal presented in this manuscript, please refer to Note 7 . Male breeding animals are introduced into cages containing virgin female animals just prior to the end of the daily light cycle. Conception status was determined by the presence of an ejacula- tory plug in the vagina of successfully mated animals. Presence of a plug is indicative (but not necessarily confi rmation) of pregnancy. Pregnant animals are separated and marked with the date of breeding and considered e0.5 of gestation. Embryo implantation nor- mally occurs between e4 and e5 in the mouse and embryos can be harvested for analysis as early as e6.5. However, later time points (beyond e9.5) may provide more discrete organs for isolation and analysis. Please note that animals more than e15 gestational age must also be independently euthanized, most commonly by decapitation. 1. Euthanize the pregnant animals using cervical dislocation, wipe the skin with 70 % ethanol, and place in a supine position. Make a superfi cial ventral incision in the skin and open the perito- neum with a second ventral incision to expose the abdominal cavity. The reproductive organs in the ventral region of the body cavity are identifi ed and the two uterine horns, oviduct and ovaries, are withdrawn from the abdominal cavity. 2. Remove the uterine horns by severing below the oviduct and dissecting it from the local mesometrium, followed by placing these in cold sterile PBS. 3. Isolate each embryo by cutting between their respective implantation sites. 4. Bluntly dissect the muscular uterine lining from the enveloped decidua tissue using fi ne forceps. 5. Gently tease apart the exposed decidua using fi ne forceps and carefully invert to release the contained embryo. The Reichert’s membrane and ectoplacental cone (trophoblast) may still be associated with the embryo, requiring additional careful dissection to isolate the embryo. 6. Image the transgenic embryos containing a fl uorescent reporter using a fl uorescent stereo dissecting microscope (such as a Zeiss Stereomicroscope SteREO Discovery V8 with LED- based illumination for GFP). This provides spatial information about the tissue-specifi c expression of the fl uorescent reporter in the developing embryo. Isolated embryonic tissues can also be independently imaged following harvesting from the intact embryo (Fig. 1 ). 7. Sever the heads and tails of isolated embryos and dissect the remaining embryonic tissue along the neural tube and ventral midline. Carefully remove the limb buds and internal organs using blunt dissection. Locate the tissue(s) of interest (identifi ed using a fl uorescent stereo dissecting microscope), carefully remove the desired material, and place it in cold PBS for further sorting or cell culture. 8. Transfer the collected organs to sterile tissue culture plates with minimal liquid and mechanically mince the tissue into fi ne pieces using sterile tweezers and scissors. Dissociate cells using enzymatic digestion (1 % (w/v) collagenase 1 prepared in serum-free media at 37 °C). The duration of enzymatic digestion can vary between different tissues and should be closely monitored throughout the process until an optimal digestion time has been established. Samples containing fully digested tissues typically appear as turbid solutions containing fi ne, granular particles. 9. Neutralize the digestion enzymes by adding serum-containing media and centrifuge samples at 1,500 × g for 4 min at 4 °C. 10. Remove the supernatant and wash the cell pellets twice with serum-containing media. 11. Resuspend the cell pellets in serum-containing media (determined by the tissue type) and seed the cells into fl asks for culture. The serum-containing media used to culture isolated cells typically contains 5.5 mM glucose, 2 mM L -glutamine, and 10 % (v/v) foetal bovine serum, but may require further supplementation depending on the cell types present. The effi ciency of digestion can be determined by visualization of isolated cells in fl asks. 12. Culture the cells in a humidifi ed 37 °C incubator (with 5 % CO 2 ) for 3–4 hr for analysis. Cells cultured for extended periods will require media replacement with fresh serum- containing media at regular intervals (alternate days to every 2 days), based on culture confl uence, cell growth, and cell proliferation characteristics. Maintain the cells until the fl ask appears confl uent, which can be determined using an inverted phase-contrast microscope. Established adherent populations from embryonic tissues typically form a confl uent monolayer in the original culture fl ask. 13. Subculture cells in confl uent fl asks using a standard technique with trypsin-EDTA as the dissociating agent. Remove the existing media and expose the confl uent cells to warm (37 °C) trypsin-EDTA for 2–3 min or until cell detachment is confi rmed through visual observation using an inverted phase- contrast microscope. Neutralize the trypsin-EDTA using serum-containing media and pellet the dislodged cells by centrifugation at 1,500 × g for 4 min at 4 °C. Resuspend the cell pellet in serum-containing media. 14. Count the cells using a hemocytometer and seed the cells at the desired cell density. Alternatively, cells should be subcultured at a predetermined subculture ratio. We typically subculture primary cells using 1:2 subculture ratios. This protocol represents an alternative step prior to cell culture, exploiting the expression of intrinsic fl uorescent reporters for isolation and quantitation of specifi c subpopulations of cells. Embryonic tissue from control and transgenic animals can be harvested and prepared as described in Subheading 3.2 ( steps 1 – 10 above). Cells are prepared and maintained in low-light conditions (avoiding direct light exposure) to preserve the fl uorescent signal. 1. Resuspend the digested cell pellets in serum-containing media (determined by the tissue type). The serum-containing media used to resuspend isolated cells typically contains 5.5 mM glucose, 2 mM L -glutamine, and 10 % (v/v) foetal bovine serum. 2. Wash the cells twice with FACS media and thoroughly resuspend the isolated cells in 200 μ L FACS media. This volume will vary based on cell density. 3. Pass the isolated cells through a 70 μ m cell strainer to remove any tissue clumps or larger cell clusters. We recommend adding propidium iodide (PI) to the cell suspension at a fi nal concentration of 1 μ g/ml to gate on live cells. 4. Acquire and characterize the isolated cells using a Becton Dickinson FACSAria or a similar fl ow cytometer. Gating can be implemented after profi ling control and transgenic cell samples based on GFP fl uorescence in FL1 channel (Fig. 2 ). 5. Collect the sorted cells in the desired serum-containing media and maintain them on ice. 6. Centrifuge the sorted cells at 1,500 × g for 4 min at 4 °C and decant the supernatant. Cells can then be resuspended in serum- containing media (described above) and seeded into fl asks for culture or dissolved in TRIzol reagent for gene expression analysis. 7. Culture the sorted cells using steps 12 – 14 of the protocol described in Subheading 3.2 (see above) or alternatively. 8. Analyze the sorted cells with respect to gene expression using the protocol described in Subheading 3.4 (see below). For transcript analysis of embryonic tissues, ...