Tissue Collection for Genetic Identification of Rodents =
Division
of Laboratory Animal Resources (DLAR)
University
of
PURPOSE:
The correct identification of the
genotype of transgenic animals in a litter is a critical aspect of research
projects using genetically modified animals.
The most common method for genotype identification involves the anal=
ysis
of DNA extracted from the tissues of the mice using the Polymerase Chain
Reaction (PCR). Sufficient DNA for PCR analysis can be obtained from a vari=
ety
of tissue sources including blood, ear punch tissue samples, tail tip biops=
ies
(tail snips), hair samples, stool, and oral swabs. Investigators are encouraged to explore=
and
use tissue collection methods that are less traumatizing than the tail biop=
sy
(tail snip) procedure whenever possible when using PCR for genotyping.
Determination of the genotype using
Southern Blot analysis requires a greater amount of tissue than the PCR
analysis procedure. The use of the=
tail
biopsy (tail snip) procedure, when properly performed, provides an adequate
tissue sample with minimal or transient pain and distress.
This document describes procedures =
and
provides standard recommendations to PIs regarding the obtainment of tissue=
for
genetic identification of mice. Th=
e text
under the section
APPLICATION:
The procedures described in this
document are applicable to colony management personnel, researchers, and
technicians who collect tissue samples for the genetic identification of
mice. The DNA prepared from tissue
obtained all of the sites (blood, ear punch, fecal samples, oral swabs, hai=
r,
and tail biopsies) is suitable for analysis by PCR. Southern Blot genotyping generally requ=
ires
the use of tail biopsy (tail snip) to obtain an adequate tissue sample.
Ear
punching does not require anesthesia in rodents when performed by a skilled=
individual. Ear punches are generally obtained at
approximately 15-17 days of age, after the ear has “thinned.” Several tissue samples can be obtained =
using
a commercially available rodent ear-punch. (http://www.harvardapparatus.com) and
the punch pattern can be used for animal identification.
The
ear punch procedure should be performed using clean gloves and a sterile
ear-punch. Manually restrain the a=
nimal
and place the punch device on the pinna of the ear (external ear) in a loca=
tion
where you want to mark the animal for identification. Press firmly to punch=
a
circular hole through the ear. As you remove the punch, be careful not to r=
ip
the delicate membrane of the pinna. Gently separate the ear from the device=
and
remove the sample for tissue sample. Bleeding after ear punching is uncommon
and the animal can be released directly into the cage.
If
the analysis of the DNA is to be performed by PCR, great care should be tak=
en
to remove all tissue from the ear punch after each animal. Sterilizing the =
ear
punch between animals using a hot-bead sterilizer will also minimize the
potential of DNA cross contamination.
Tissue for PCR genetic analysis will be obtained =
from
ear punch samples. 2 mm ear pu=
nches
will be obtained from animals at 15 days of age or older. Animals will be manually restrained=
and
the sample collected aseptically.
Animal identification will be via a standard animal identificati=
on
ear punch pattern.
Obtaining
tissue from a mouse or rat for DNA analysis via tail biopsy is a safe,
effective and humane procedure. When performed properly it causes only mini=
mal
or transient pain and distress, and induces no more “physiological impact”
(change in heart rate, body temperature, or activity level) than just
restraining the animal for the procedure. DNA prepared from tail biopsies is
suitable for analysis by either Southern Blot or PCR.
The
removal of tail tip of a mouse involves cutting skin, nervous tissue,
vasculature and developing vertebrae. This procedure, while generally
considered a minor procedure, carries a potential for acute and chronic pai=
n.
Careful consideration must be given to the timing of this procedure relativ=
e to
the animal’s age and the length of tail tissue removed. A review of the current literature (cit=
ed at
the end of this document) will show that definitive guidelines have not been
established concerning animal age at biopsy, length of biopsy tail tissue or
anesthesia/analgesia. Alternative=
s to
tail biopsy involving the use of less invasive and more humane methods such=
as
ear punches, blood samples, hair samples, stool, mucous epithelial cells or
saliva swabs should be initially investigated prior to electing tail biopsy=
as
the method for tissue collection for genetic analysis. =
=
Animals
should be between 10 and 21 da=
ys
old. At this age, the tail tissue =
is still
soft, the tail vertebrae have not yet calcified, and the yield of DNA is the
highest. Prompt analysis allows mi=
ce to
be genotyped prior to weaning, facilitating efficient cage use and housing =
cost
savings.
Anesthesia:
For
mice and rats 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 isopr=
opyl
or ethyl alcohol for 10 seconds [Ca=
ution: ethyl chloride, isopropyl alcohol, and =
ethyl
alcohol are all flammable] or by the application of ethyl chloride (=
ht=
tp://www.gebauerco.com/Default.asp)
[see below for additional information]=
. Investigators may wish to consider the =
use of
general anesthesia as an alternative to local anesthesia in specific cases.=
For
mice and rats greater than 21 days of age: The use of a
local or general anesthetic is required prior to collection of tissue.
For
rats greater than 35 days of age: The use of a general anesthetic is
required.
Recommended Tail Biopsy Anesthesia
Regimens:
Local: Ethyl Chloride spray
Ice-cold Alcohol (isopropyl or ethyl)
Inhalant: =
Isoflurane
1-4%
Injectable: Ketamine/medetomidine (mouse 75-100
mg/kg IP/1 mg/kg IP; rat 60-75 mg/kg IP/0.5 mg/kg IP) OR Ketamine/xylazine
(mouse 90-120 mg/kg IP/10 mg/kg IP; rat 40-80 mg/kg IP/10 mg/kg IP)
Ketamine/medetomidine is recommended as the injectable of choice due to the
much more rapid recovery of the rodent)
The
tail biopsy procedure must be performed using clean gloves and a sterile sh=
arp
scalpel, scissors, or razor blade. Tail
skin should be disinfected with alcohol prior to incising the tip (Do not u=
se
iodine solutions because they may interfere with DNA analysis.) Manually
restrain the mouse or rat 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.). Make one clean cut through the tail. If the analysis of the DNA is to be per=
formed
by PCR, great care should be taken to remove all tissue from the scissors or
scalpel after each animal. Sterilize the scalpel, scissors, or razor blade
between animals. If a scalpel or razor blade is used, also disinfect the wo=
rk
surface on which the tail is placed between animals.
The tissue taken should be as small=
as
possible with 5 mm the maximum len=
gth
of tail tip removed (skin can be pushed down toward the tip of the tail so =
that
the vertebrae are avoided). Tail b=
iopsy
samples of greater than 5 mm in length will most likely damage the vertebrae
and are not recommended. If the pr=
oper
procedures are followed, the yield of DNA from 5 mm of tail should exceed 50
micrograms, enough for multiple analyses.
The amount of DNA obtained does not proportionally increase with sam=
ples
of over 5 mm length and, in most cases; a 2 mm tail biopsy is more than
adequate.
Following the biopsy procedure,
bleeding should be controlled using local pressure and sterile gauze. The use of a cotton-tipped applicator w=
ith 1%
lidocaine with epinephrine may enhance hemostasis as well as providing
post-procedural local analgesia. A=
fter
releasing the animal back into the cage, it should be observed to make cert=
ain
that the bleeding has stopped. The
presence of blood in the cage may cause aggression between cagemates. It may be necessary to use styptic powd=
er,
silver nitrate, or surgical glue in cases where bleeding is not controlled =
with
digital pressure.
Tail biopsies are done on rodents 10-21 days of
age. The animal will be manual=
ly restrained,
the tail tip cleaned with alcohol and then ethyl chloride spray applied
until the tail is blanched. The tail biopsy procedure will be performed
using clean gloves and a sterile sharp scalpel, scissors, or razor blad=
e.
Tissue taken will be as small as possible and not exceed 5mm in
length. One clean cut will be =
made
through the tail. Any bleeding=
will
be controlled with pressure and sterile gauze initially followed by the=
use
of 1% lidocaine with epinephrine, silver nitrate, or styptic powder sho=
uld
such be necessary to control the bleeding. Instruments will be steriliz=
ed
with a bead sterilizer between animals.
The
use of hair bulbs in human forensic medicine is common and sufficient DNA c=
an
be extracted from a single hair bulb to perform genetic screening by PCR.
The
actual sample collection procedure requires the manual restraint of the ani=
mal
and the removal of a tuft of hair from the ventral body using forceps. While there is no necessity for sterile
instruments, the use of a new set of forceps for each animal minimizes the
potential of cross contamination between animals.
Hair
bulb sampling can be performed at any age and does not require any anesthes=
ia
or analgesia. There are a number of
commercially available DNA extraction kits for hair bulb samples.
Tissue for genetic evaluation will be obtained fr=
om
hair bulbs. The animal will be manually restrained and a small tuft of =
hair
removed from the ventral abdomen using clean forceps. The animal will then be returned to=
the
cage.
Feces contain sloughed intestinal
epithelial cells which may serve as a source of DNA for genetic analysis.
Saliva sampling is routinely used f=
or
genetic analysis in humans to determine paternity. A small amount of saliva contains suffi=
cient
oral epithelial cells to permit PCR analysis.
Unlike the human situation where swabs are used to collect the
epithelial cells, the collection technique in rodents involves the washing =
of
the oral cavity of weanling animals using a plastic pipet tip (Irwin, et.al.)
1. <=
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/span>Dennis, MB. IACUC Review of Genetic Engineering.=
Lab
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Transgenic Mice by PCR Analysis of Saliva. Nat Biotechnol 1996 Sep;14(9):
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/span>Schmitteckert EM, Prokop CM and Hedrich HJ. DNA
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Health. BioTechniques 1994; 17:1030-1032.
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10. Shinohara H. The Musculature of the Mouse Tail is
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and Distress in Laboratory Rodents and Lagomorphs. Report of the Federation of European
Laboratory Animal Science Association Working Group on Pain and Distress.
Laboratory Animals (28): 97-112.
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Nov 47(6):10-18. 15. Williams WO,
Riskin DK, Mott KM. 2008. Ul=
trasonic
Sound as an Indicator of Acute Pain in Laboratory Mice. Journal of the American Association of
Laboratory Animal Science 47 (1):8-10. 16. DeMarco G ,Pascoe P, Meyer R, Fish R, Brunson D,
Heavner J, Cooper D. 2008. Anatomy,
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Genotyping of Mice and Rats, ARAC Guidelines, Office of Animal Care and Use,
National Institutes of Health, Revised 9/12/07 (http:/=
/oacu.od.nih.gov/ARAC/documents/Rodent_Genotyping.pdf) 18. Boston University http://www.bu.edu/research/compli=
ance/oversight-committee/iacuc/policies/mouse-tail-biopsy-guidelines.shtml<=
/a> 19. University
of Minnesota http://ww=
w.ahc.umn.edu/rar/tailsnip.html 20. Indiana University S=
chool
of Medicine http://www.iupui.edu/~somiacu=
c/worddoc/Tail%20snip%20guidelines%20Feb%2003.doc Using Ethyl Chloride =
for
Local Hypothermic Anesthesia: Ethyl chloride is a topical aer=
osol
anesthetic skin refrigerant. When topically applied to the skin, Ethyl =
chloride
creates an instantaneous cooling effect on the surface of the site by
immediate evaporation of the product. The coldness created by the spray
interrupts the body’s ability to experience pain. Ethyl chloride should be the la=
st step
before performing the tail biopsy, after the area has been appropriately
disinfected and the instruments are ready. Spray ethyl chloride on the =
tip
of the tail for 3 to 7 seconds or until the skin begins to blanch. When=
the
skin begins to turn white, the numbing has taken effect and it is time =
to
stop spraying. Do not frost the skin. The anesthetic effect will last u=
p to
60 seconds. For more information see http://www.gebauerco.com/Default.asp?strAction=3DEthylChloride
Veterinary Recommendations—Genetic Identification of Rodents=