2.1 Collection of embryonic glands

The age of the embryo is determined by checking vaginal plugs. Noon of the day after copulation is counted as day 0.5. All procedures are performed under sterile conditions.

• Remove uterus with embryos and transfer to 10 cm Petri dish with PBS
• Carefully remove embryos from the uterus with forceps
• Cut off the head of the embryo with dissecting knives
• Transfer trunks to fresh Petri dish using forceps. If the genotype of the mutant embryos cannot be determined by morphological criteria it is advisable to transfer each embryo into one well of a 6 well plate filled with 3 ml of dissection medium. In order to reduce confusion it is best to dissect the embryo on one side of the dish and set up the culture in the opposite well
• Turn embryo on its belly and with dissecting knives cut in half along the middle of the spinal cord
• Reserve small piece (e.g. tail) for PCR genotyping of embryo
• Turn embryo skin side down and remove inner organs
• Locate gonads
  
• determine sex of the embryo: testes are recognized by the presence of testis cords, ovaries lack any morphological distinction. Use only female embryos
  
• Peel off the skin with dissecting knives starting from the back towards the belly. The glands will be visible as small circles surrounded by a slightly darker ring of dense mammary mesenchyme
  
• Cut out individual mammary anlagen
  
• Collect mesenchyme from the flanks by lifting the tissue overlying the rib cage

2.2 Culture of embryonic glands

Since the individual anlagen are too small to transplant, 2 to 3 glands are combined on a piece of dorsal mesenchyme and cultured overnight up to 2 days. The tissues will merge and form an aggregate that can be manipulated for transplantation. The culture period also leaves time to perform genotyping analyses by PCR

• Prepare one culture dish per embryo by placing a stainless steel grid in a 35mm petri dish (or on the opposite side of the 6 well dish in which the dissection was performed). Place a circle of nulepore filter over the center hole of the grid. Fill the dish with PBS so the filter touches the liquid surface and is kept moist during the assembly procedure.
  
• Place 2 pieces of mesenchyme apart from each other on the filter.
• Place two to three mammary buds on top of one piece of mesenchyme with a transfer pipet.
  
• When all the cultures are assembled replace the PBS with culure medium (DMEM/Ham’s F12 with 10% FCS, glutamine and penicillin/streptomycin) and incubate at 37^oC in a humidified atmosphere containing 5% CO₂.

2.3 Transplantation into cleared fat pad

After a culture period of approximately 20 to 36 hours the cultures are transplanted into the cleared fat pad of a syngenic or immune compromised hosts. (Instructions for clearing of the fat pad). It is recommended that you reserve the cleared portion of the fat pad on a slide, fix it and process by whole mount staining. Mark each mouse with ear marks so that incompletely cleared hosts can be identified after staining

• Carefully submerge nuclepore filter with cultures in trypan blue for a few minutes
• Rinse filters in PBS. The stain helps in visualizing the small piece of tissue to be transferred
• With the micro dissecting tweezers poke a pouch in the center of the cleared fat pad. Pick up one culture at a time and insert into the pouch. Sometimes the cultures tend to get destroyed during this procedure and the yield of successful transplants can be as low as 30 to 50%
• Close incision of the skin with wound clips
• Watch mouse until recovered from surgery