Identification of Pre-weanling 7-Day-Old Mice with Pinna Edge Biopsies

The scope of this research is to discover, develop, and then verify a novel method of simultaneous identification and genotyping for pups between the ages of 7 and 14 days old. This protocol demonstrates a novel method to identify and genotype mice at 7 days old, enabling earlier research access than current methods typically performed at 14 days or later. The current PEB method provides nine ID patterns, sufficient for litters up to nine pups.

For larger litters, new PEB combinations must be developed to expand identification options. To begin, gently restrain a mouse pup in a position that permits visualization and access to both ears. Using the non-dominant hand, pick up the pup by the base of the tail and place it on a stable surface on top of the cage bars.

Use the thumb and forefinger of the non-dominant hand to apply gentle pressure along the pup's back until the loose skin between the ears can be pinched. Then pinch the skin securely to restrict head movement, ensuring the animal breathes normally. If restraining with the left hand, orient the pup so that its nose points away from the handler's body, making the right ear pinna accessible.

To access the left ear pinna, rotate the restraining hand so that the left side of the pup faces the handler. If restraining with the right hand, orient the pup's nose away from the handler to expose the left ear pinna. To access the right ear pinna, supinate the hand grasping the animal until the right side of the pup faces the handler.

Use a total of nine patterns for the pinna edge biopsy, or PEB, which includes eight biopsy patterns and one no biopsy option. Use clean, small dissection scissors, or McPherson-Vannas scissors to perform each biopsy. Disinfect the instruments by either spraying them with disinfectant and/or 70%isopropyl alcohol or wiping with alcohol wipes after every use.

Hold the scissors using the dominant hand. To create a vertical PEB, trim the lateral edge of the ear vertically, removing approximately one-third of the pinna. To create a horizontal PEB trim the dorsal edge of the pinna at a 45-degree angle, removing about one-third of the pinna.

Create one of eight designated PEB patterns. Use fine forceps to carefully place the excised tissue into a sample container for genotyping. If bleeding occurs at the biopsy site, apply gentle pressure using clean gauze or a cotton tip applicator while the animal remains restrained.

Gently return the pup to its cage with the dam. Then observe the animal for one to two minutes to check for continued bleeding from the biopsy site. Pinna biopsy patterns remained visibly identifiable up to postnatal day 63 in mice initially marked on postnatal day 7.

The right horizontal pinna biopsy created at a 45-degree dorsal angle appeared more horizontal by postnatal day 63 as the ear matured. Both male and female mice showed a significant increase in body weight from postnatal day 7 to 63, with males weighing more than females from postnatal day 30 onwards.