Tail Biopsy. The proper identification of transgenic
animals in a litter is critical to the efficient pursuit of research
and in reducing the number of animals involved in a research project.
Most often the genotype is determined by analysis of DNA extracted
from tissues of young mice. Analysis by the Polymerase Chain Reaction
(PCR) requires the least amount of DNA. DNA for PCR analysis can be
obtained from ear punches, hair samples, or oral swabs (see references
1-6). Depending on the requirements of the study, investigators are
urged to consider these alternatives. Larger amounts of DNA are required
for Southern Blot determination of the genotype. The IACUC has determined
that obtaining tissue from a mouse for DNA analysis via tail biopsy
is a safe, effective and humane procedure that causes minimal or transient
pain and distress when performed properly. DNA prepared from tail
biopsies is suitable for analysis by either Southern Blot or PCR.
1. Procedures for tail biopsy for DNA analysis and/or
genotyping must be described in an approved animal
protocol.
2. Ideally, mice should be 10-21 days old. At this
age, the tail tissue is soft (vertebra are not yet
calcified) and the yield of DNA is highest. In addition,
prompt analysis of tail tissue allows the desired mice
to be identified prior to weaning which can facilitate
more efficient use of cage space.
a. For mice 10-21 days of age: Because pain sensory
development may be complete, and to further minimize
any transient pain or distress, investigators are strongly
encouraged to apply local anesthesia to the tail. Local
anesthesia may be achieved by immersion of the tail
in ice cold ethanol for 10 seconds. Alternatively,
the tail can be disinfected with 70% ethanol and allowed
to dry, followed by an application of ethyl chloride
spray or other suitable anesthetic as recommended by
the attending veterinarian.
b. For mice greater than 21 days of age: The use of
a general anesthetic is required prior to collection
of tissue. Avertin (tribromoethanol) can be used in
mice by intraperitoneal injection of 0.3-0.6 mg/g.
Other anesthetic agents for use in mice can be found
on this web site at Anesthesia
and Analgesia for Mice.
3. Manually restrain the mouse between thumb and forefinger.
This is a convenient time to identify the animals using
the appropriate method (i.e. ear punch, ear tag, transponder
etc.).
4. With sterile scalpel, razor blade, or scissors
cleanly excise the distal 5 mm of tail. If the proper
procedures are followed, the yield of DNA from 5 mm
of tail should exceed 50 micrograms, enough for multiple
analyses. The yield of DNA does not proportionally
increase as tail fragments larger than 5mm are used.
If small amounts of DNA are required, investigators
should consider taking only 2 mm of tail. If the analysis
of the DNA is to be performed by PCR, great care should
be taken to remove all tissue from the scissors or
scalpel after each animal. Disinfect the scalpel or
scissors between animals. If a scalpel is used, also
disinfect the work surface on which the tail is placed
between animals.
5. The investigator must monitor the animals to assure
hemostasis after the animals are returned to the cage.
Apply digital pressure, silver nitrate, or other means
of hemostasis.
6. Repeat tail biopsies require general anesthesia
and must be justified in the protocol.
Template
Tails are collected from 10-21 day old mice without
general anesthesia. The mouse is gently but firmly
grasped by skin behind the neck. To provide local anesthesia,
the tail is dipped in ice cold ethanol for 10 seconds
before performing the tail biopsy. A sterile scalpel,
razor blade, or scissors is used to cleanly excise
the distal 5 mm of the tail. Silver nitrate or direct
pressure is used on the tip of the tail to effect hemostasis
and the animal is returned to the cage once hemostasis
is verified. Mice older than 21 days are anesthetized
with Avertin before the tail biopsy is performed. Depth
of anesthesia is evaluated by the toe pinch technique
before the biopsy is collected from anesthetized animals.
References
1. Hofstetter JR, Zhang A, Mayeda AR, Guscar, T,
Numberger JI and Lahiri DK. Genomic DNA from Mice:
A Comparison of Recovery Methods and Tissue Sources.
Biochem Mol Med 1997 Dec; 62(2):197-202.
2. Dennis, MB. IACUC Review of Genetic Engineering.
Lab Animal 2000 Mar; 29(3):34-37.
3. Irwin MH, Moffatt RJ and Pinkert CA. Identification
of Transgenic Mice by PCR Analysis of Saliva. Nat Biotechnol
1996 Sep;14(9): 1146-8.
4. Schmitteckert EM, Prokop CM and Hedrich HJ. DNA
Detection in Hair of Transgenic Mice - A Simple Technique
Minimizing the Distress on the Animals. Laboratory
Animals 1999; 33/4: 385-389.
5. Couse JF, Davis VL, Tally WC and Korach KS. An
Improved Method of Genomic DNA Extraction for Screening
Transgenic Mice. National Institute of Environmental
Health Sciences, National Institutes of Health. BioTechniques
1994; 17:1030-1032.
6. Malumbres M, Mangues R, Ferrer N, Lu S and Pellicer
A. Isolation of High Molecular Weight DNA for Reliable
Genotyping of Transgenic Mice. BioTechniques 1997;
22/6:1114-1119.
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