UNIVERSITY OF COLORADO DENVER
STANDARD PROTOCOL FOR REDERIVATION OF MOUSE COLONIES: IMPLANTATION OF
CRYOPRESERVED OR FRESH EMBRYOS
Note: Complete this protocol form only if you are having a mouse line rederived by the UC Denver Transgenic and Gene Targeting
Core laboratory (TGTC). Any manipulations you plan to perform at UC Denver on the mouse line produced under this protocol must
be submitted online in a separate, complete, experimental protocol application form.
Please complete all the sections which are presented in green
SECTION A - ADMINISTRATIVE INFORMATION
1) PRINCIPAL INVESTIGATOR (PI) NAME:
a. DEPARTMENT:
b. CONTACT INFORMATION FOR PI: TELEPHONE:
c.
PROTOCOL AFFILIATIONS:
MAIL BOX:
UC Denver
Barbara Davis
Denver Health
Other
EMAIL:
Cancer Center
2) CAMPUS/FACILITY WHERE RESEARCH IS CONDUCTED:
ANSCHUTZ MEDICAL CAMPUS
OTHER:
a. Location of Laboratory
P15BUILDING 0384 ROOM NUMBER (if using Transgenic Core)
b. Location Of Area Where Animal Procedures Are Performed
P18 BUILDING 0410B ROOM NUMBER (if using Transgenic Core)
3) PROTOCOL TITLE: REDERIVATION OF MOUSE COLONIES: IMPLANTATION OF
CRYOPRESERVED OR FRESH EMBRYOS
TYPE OF PROTOCOL:
NEW
PILOT
REPLACEMENT FOR #:
4) PROTOCOL ASSOCIATES
a. INVOLVED IN ANIMAL PROCEDURES : (if using Transgenic Core) Abby Zamora, Saiphone Webb, Peter Koch
(PQ forms on file)
b. CONTACT PERSON:
TELEPHONE:
MAIL BOX:
EMAIL:
5) TOTAL NUMBER OF EACH SPECIES IN APPLICATION: Mouse (See Section B.5. for Experimental Groups
and Numbers Tables) =
6) GRANT/PROJECT TITLE:
a.
FUNDING AGENCY/SOURCE: (Projects funded with internal support require departmental chair approval letter)
NIH
NON PROFIT AGENCY (include name)
SBIR
TTO-POC(Tech Transfer Office
FOR PROFIT COMPANY (include name)
OTHER (include name)
b. GRANT NUMBER/ID (If known):
IN REVIEW, DATE:
c.
(Requires departmental chair approval letter)
MOU Attached
OGC ROUTING NUMBER:
AWARDED, DATE:
SPEEDTYPE NUMBER:
7) INCLUDED IN THE APPENDIX (select all that apply):
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 1 of 13
DEPARTMENTAL APPROVAL LETTER
PERSONNEL QUALIFICATION FORMS(please complete and SIGN a PQF for PI)
SECTION A2 – INDIVIDUAL INVESTIGATOR SPECIFIC PROJECT INFORMATION
1.
What specific strain(s) and/or transgenic line(s) will be expanded or rederived?
2.
Why is the line(s) being expanded or rederived?
3.
4.
Describe any special features/characteristics of the line/strain, especially those that pertain to the animal’s
health, longevity and abnormal phenotype.
POTENTIAL CONTRIBUTION(S) TO BIOLOGY AND/OR HUMAN MEDICINE:
These procedures may help produce more rapid and accurate accumulations of experimental results
which may benefit development of new medicines and may contribute to elucidation of the biochemical
processes of living organisms.
(Please add a statement of the potential contribution to biology and/or human medicine for your particular line)
5,
Source of embryos for rederivation
Cryopreserved (in-house, please provide collection/cryopreservation number)
Cryopreserved (imported)
Fresh (please provide protocol number)
6.
Will the mice produced by the colony expansion/rederivation procedures be housed and/or used at UC Denver?
Yes. If yes, what is the number of the protocol that describes the use of the mice?
No. If no, where (at what institution) will the mice be housed/used?
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 2 of 13
SECTION B - PROJECT DESCRIPTION
1.
PROJECT GOALS IN NON-SCIENTIFIC (i.e. “LAY”) LANGUAGE
The goal of this study is to rederive a mouse strain (breed) using fresh or cryopreserved embryos for surgical
implantation (Rederivation) for one of the following reasons:
a) Produce pathogen-free animals from a compromised mouse strain (breed) or colony.
b) Rapidly produce larger numbers of animals more quickly than normal mating. This process is termed
speed expansion or colony expansion.
c) Line/colony rescue
Mouse pathogens can give rise to a variety of problems in the experimental colony. Immunologic studies may be
influenced by circulating antibodies. Fecundity (fertility or ability to reproduce) may be adversely affected
threatening loss of valuable transgenic and other research colonies.
Rederivation is an effective technique to ’clean up’ or remove the majority of mouse pathogens and
ectoparasites from a mouse colony. Since most pathogens are spread via fecal or oral contact, surgical transfer
of fertile embryos into SPF (specific pathogen free) foster mothers can eliminate the majority of pathogens.
Colony speed expansion (CSE) is a valuable tool as the name implies, to expand the size of an existing colony
within a shorter time that could be achieved through normal mating. This is of particular importance when
experimental groups of age-matched animal must be obtained and used for a specific experimental protocol.
Similarly, a colony that is not reproducing effectively (diminished fecundity) is at risk of being lost. Poor
reproductive performance can be due to numerous factors including the strain of animals that make up the
colony, characteristics imparted by the presence of the transgene, age of the colony etc. It is possible to “rescue”
these lines by producing new offspring using rederivation.
(Individual Investigators must provide specific information about their strain or line in section A2)
2. NARRATIVE DESCRIPTION OF ANIMAL PROCEDURES:
There are two primary methods of producing embryos for rederivation or colony expansion:
a.
.
Non-transgenic donors obtained from vendors.
Non-transgenic embryo donors are used when the resulting embryos are not required to be or are preferred
to be non-homozygous. This is a short procedure and can usually be completed within one week.
The investigator must have available at least six sexually mature stud males of the selected strain or
transgenic line from their colonies. The Transgenics Core (TGTC) will purchase an equal number of nontransgenic embryo donor females of the same strain from commercial vendors. The donor females will be
housed in the TGTC mouse rooms for superovulation.
While in the transgenic core, the embryo donor females are superovulated by IP injection of PMSG
(pregnant mares' serum gonadotropins; 5 IU; 0.1 ml; IP) at 2 PM on day one. An IP injection of hCG (human
chorionic gonadotropins; 2.5-5 IU; 0.1 ml; IP) is administered 46 hours later (day three) at 12 PM.
Following the hCG injections, the investigator’s lab personnel must pick up the hormonally primed female
donors from the TGTC by 1 or 2 PM. The donors will be taken to the investigator’s colony room where they
are mated overnight with the selected transgenic stud males. The following morning (day 4), the lab
personnel must bring the mated donor females to the TGTC for embryo harvest by 10 AM.
The donors are euthanized by C02 narcosis (followed by cervical dislocation) and the embryos are collected,
cleaned and surgically transferred to pseudopregnant females.
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 3 of 13
b.
Transgenic embryo donors produced by the investigator:
Background strain considerations or genetic background may dictate that this method be used. Usually, the
investigator is the sole source of the particular strain or genetically altered animals to be expanded.
Transgenic embryo donors are necessary when homozygous or a genetic background that will produce as
many homozygous embryos as possible is preferred. Many transgenic lines are produced in-house and are
not commercially available. This technique may require approximately nine weeks until embryos transplant
surgeries are complete.
1. This requires that the investigator’s lab produces the transgenic embryo donors from their colony. Three
transgenic males are mated with three transgenic females. After one week, the females are separated and
allowed to give birth. Male pups are culled at approximately one week of age. At 21 days of age, the
female pups are collected to be used as the transgenic embryo donors. It will take about 42-49 days to
generate the embryo donors using this method.
Investigator’s lab personnel will administer both superovulation hormones to the donors and set up matings
with their transgenic breeder males as described above. Each donor will be mated to one of the available
transgenic stud males. The following morning, the mated donor females must be delivered to the TGTC by
10AM for embryo collection.
In the TGTC, the donor females are euthanized by C02 narcosis followed by cervical dislocation, the
embryos collected and washed in 5 changes of embryo culture media and transferred to pseudopregnant
recipient mothers.
c. Embryo transfer from cryopreserved embryos
If cryopreserved embryos from the desired lines are available, these may be used as follows:
The embryos are thawed by our standard cryopreservation protocol. Thawed embryos are recovered and
washed through five changes of embryo media to complete the cleaning process (described below) begun
prior to cryopreservation. Embryos can now be surgically transferred as described for fresh embryos.
Cleaning process: Four rinses in 1 ml each using sterile culture media for each change prior to
cryopreservation. Upon recovery of the embryos, complete cleaning process by passing embryos through
five 1 ml rinses of sterile culture media.
A. Pseudopregnancy - (PsPg) is stimulated by mating 10 mature female mice from our standing recipient
colony, for each day of anticipated embryo transfers, with males from the Transgenic Laboratory's standing
vasectomized male colony. Those females with a vaginal plug the next morning will be the recipients for the
embryo transfers. (This usually produces 4-6 plugged females. The non-plugged females along with those
not used for embryo transfer are placed back in the TGTC's colony.)
B. Embryo Transfer- The embryos are transferred to the oviducts of the PsPg recipient females as described
in Appendix A. The transferred females will be housed in a VAF (viral antibody free) colony within the
TGTC until birth and weaning of the putative transgenic mice. Following weaning, tail biopsies are taken
and the animals transferred to the investigator.
One or more of the following procedures may be used:
Tail biopsies for DNA Analysis: Standard procedure for mice between 3-4 weeks of age, one tail sample
(~0.2 cm) is allowed without anesthesia or ligature. Very little bleeding occurs as the tail tip is cartilage and
is not ossified. Older mice are anesthetized and the tail is ligated with #1 silk thread about 1.0-1.5 mm
from the tip. The tail is clipped with scissors immediately distal to the ligature and the mouse placed back
into its home cage to recover. Alternatively, the tail is cauterized with a cautery pen.
Ear Biopsies for DNA Analysis: The mouse is restrained by hand and an ear punch biopsy is taken or a
small snip (2.0mm) of pinna periphery is excised with scissors. No analgesia is necessary for ear biopsies.
There is no bleeding and pain is momentary similar to ear tagging (or ear punching) for identification.
Blood samples: Blood samples are rarely used but if done, will be taken from isoflurane-anaesthetized
mice by one of two methods. For amounts of 0.25 ml, submandibular blood sampling is used. For lesser
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 4 of 13
amounts (0.1 ml), the lateral tail vein will be nicked and the blood collected.
The foster mother and non-transgenic pups will be sacrificed by CO2 narcosis (followed by cervical
dislocation) and the positive pups transferred to the investigator's appropriate IACUC “use” protocol.
3.
STUDY END-POINT FOR ANIMALS (Select all that apply)
a. Animals will not be euthanized at study completion.
b. Animals will be euthanized:
c. without significant treatments or experimental procedures.
d. after defined period of time: embryo donors:3-4 weeks; recipient females: 28 days post partum
4.
ANIMAL IDENTIFICATION:
a.
Species: Mouse
b.
Strain (for rodents): as in section A 2.1
c.
Sex: male/female
d.
Age and/or weight range: 3 weeks – 1 year
5.
EXPERIMENTAL GROUPS AND NUMBERS: Stress Category D
a. Tables:
A1: vendor supplied embryo donors
Experimental/Control Group Number of Animals per
Names
Group
Embryo donors purchased
from vendor
Number of Repetitions
(per group)
Total number used
in group (sum per row)
6-10
2
20
6-10
2
20
Recipient females
5
2
10
Vx males
5
2
10
Breeder males (investigator)
A2: Investigator supplied embryo donors
Experimental/Control Group Number of Animals per
Names
Group
Embryo donors provided by
investigator
Number of Repetitions
(per group)
Total number used
in group (sum per row)
6-10
2
20
Recipient females
5
2
10
Vx males
5
2
10
A3: Regeneration using existing frozen embryos
Experimental/Control Group Number of Animals per
Names
Group
Number of Repetitions
(per group)
Total number used
in group (sum per row)
Recipient females
5
2
10
Vx males
5
2
10
b.
Number of animals required for the entire study:
A1: Vendor supplied embryo donors:
up to 60 x
# lines = Total number________
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 5 of 13
A2: Investigator supplied embryo donors: up to 40
x # lines = Total number________
A3: Regeneration using existing frozen embryos: up to 20 x
c.
# lines = Total number _________
How was the number of animals in each group determined?
Each group represents the number of animals required to produce sufficient embryos necessary for
successful regeneration. See following notes:
i. Embryo donors will either be ordered from vendors or produced by mating with two or more of the
males provided by Investigator. The exact source of the donors depends upon the requirements of
the Investigator's transgenic mouse line and strain.
ii. Transgenic females produced within the investigator’s colony may be required to produce litters
from which to draw up to from six to ten 21-day old donor females.
iii. In either of the above (i and ii) schemes for obtaining the embryo donors, six to ten males in
Investigator's colony of transgenic animals will be used to mate with the embryo donors.
iv. The estimated number of recipients represents the minimal number of animals necessary to support
successful regeneration.
d. How was the number of repetitions for each group determined?
Occasionally, the females don’t produce sufficient numbers of embryos or fertilization of embryos is
insufficient and the procedure may have to be repeated.
6. EXPERIMENTAL AGENTS:
Experimental Agents
PMSG
Frequency of Duration of Treatment
Dosing
Effect
Dose
Route)
Volume
5 IU
IP
0.1 ml
Single Dose
48 hours
2.5-5 IU
IP
0.1 ml
Single Dose
12 hours
HCG
7.
NEUROMUSCULAR BLOCKING AGENTS (I.E., PARALYTICS): N/A
8.
HAZARDOUS MATERIAL USE: PMSG and hCG
SECTION C - ANESTHESIA AND ANALGESIA
1. PRE-ANESTHETIC/ANALGESIC OR SEDATIVE DRUGS: analgesic: Banamine, 2.5 mg/kg immed. prior to
surgery
Drug
Banamine
Dose
Route)
Volume
2.5 mg/Kg
SC
0.05 cc
Frequency of Duration of Treatment
Dosing
Effect
Single Dose prior
12 hours
to surgery
e. ANESTHETIC DRUGS :
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 6 of 13
Drug
Initial Dose
Supplemental
Dose
Route
Isoflurane
5%
3-3.5%
Inhalation
For Which
Procedure?
Laparotomy
3.
ANESTHESIA MONITORING (CHECK ALL THAT APPLY):
b. Response to toe/skin pinch.
4.
FREQUENCY OF MONITORING:
b. Every 2-3 min.
5.
POST PROCEDURAL ANALGESIC OR TRANQUILIZING DRUGS:
DRUG
Banamine Analgesic
DOSE
ROUTE
2.5 mg/Kg
SC
Expected
Time Under
Anesthesia
FREQUENCY
OF DOSING
once every 12
hours post
surgery
15 min
DURATION
OF
TREATMENT
WHICH ANIMALS?
48 - 72 hrs
Laparotomy
SECTION D - POTENTIAL HEALTH CHANGES
1. POTENTIAL HEALTH CHANGES
a. Decreased food and water intake.
b. Abscesses.
c. Dehydration.
d. Infection
e. Malnutrition.
f.
General weakness.
g. Diarrhea.
h. Constipation or ileus.
i.
Seizures.
s. Weight loss, specify as a % of total body weight:
t. Hyper/hypo-glycemia. Explain:
u. High incidence of carcinogenesis. Explain:
v. Behavioral changes. Explain:
w. Other. Specify:
x. None
j.
k.
l.
m.
n.
o.
p.
q.
r.
Coma.
Labored breathing.
Hypothermia.
Hyperthermia.
Skin abnormalities.
Paralysis.
Ataxia.
Urinary incontinence.
Excessive urination.
2. MONITORING FOR ANTICIPATED HEALTH CHANGES (Check all that apply):
Observed/Assessed Parameter
Frequency of Assessment
a. Food/water consumption
/ (# / day, week, or month)
b. Body weight
/ (# / day, week, or month)
c. Pain or discomfort
1/day (# / day, week, or month)
d. Signs of infection
1/day (# / day, week, or month)
(redness, swelling, discharge or dehiscence)
e. Behavior, activity, or posture.
1/day (# / day, week, or month)
f. Blood glucose
/ (# / day, week, or month)
g. Tumor growth
/ (# / day, week, or month)
h. Other:
/ (# / day, week, or month)
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 7 of 13
i.
Not applicable.
3. CRITERIA FOR PREMATURE REMOVAL FROM STUDY (Check all that apply):
a. Inability to eat or drink adequately.
b. Weight loss more than 15% of body weight.
c. Excessive generalized or localized pain and/or discomfort.
d. Uncontrollable infection, sepsis.
e. Markedly reduced response to stimuli or inability to ambulate properly.
f. Chronic hyper/hypo-glycemia; specify criteria:
g. Excessive tumor burden (>2cm length or >10% of total body weight)
Specify and justify alternative criteria:
h. Veterinarian’s discretion based upon humane issues. (required)
i. Other. Specify:
4. ANIMALS REMOVED FROM THE STUDY WILL BE:
a.
Euthanized.
SECTION E - RESTRAINT, DEPRIVATION, AND EUTHANASIA
1. RESTRAINT:
a.
Routine.
2. SPECIAL HOUSING, CONDITIONING, DIET OR OTHER CONDITIONS:
a.
None Apply.
b.
Yes. The following apply (Check all that apply):
(1) Prolonged exposure to high or low temperatures.
(2) Prolonged exposure to non-standard humidity.
(3) Prolonged exposure to non-standard atmosphere.
(4) Non-standard housing.
(5) Prolonged exposure to non-standard light cycle.
(6) Water restriction for longer than 12 hours.
(7) Food restriction for longer than 24 hours (simple stomach animals) or
longer than 48 hours (ruminants).
(8) Specialized or purified diet (include diet formulation datasheet in the
appendix).
(9) Other. Specify:
c.
If yes, justify the special conditions:
3. METHOD OF EUTHANASIA (check all that apply):
f.
Carbon dioxide exposure (followed by cervical dislocation)
SECTION F - ANIMAL ORDERING AND HOUSING INFORMATION
1. SOURCE (check all that apply):
a.
Purchased from an approved vendor.
f.
Other. Specific details on strains and protocol source in section A2
2. ANIMAL HOUSING (Check all that apply):
UC Denver Center for Comparative Medicine
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 8 of 13
3. WILL ANIMALS WILL BE REMOVED FROM THE ANIMAL HOUSING FACILITY?
a. No.
SECTION G - RATIONALE FOR USE OF ANIMALS
AND APPROPRIATENESS OF THE SPECIES AND NUMBERS USED
1.
LIVING ANIMALS ARE REQUIRED FOR THIS STUDY BECAUSE:
a. The complexity of the processes being studied cannot be duplicated or modeled in
simpler systems.
b. There is not enough information known about the processes being studied to design
nonliving models.
c. Preclinical studies in living animals are necessary prior to human testing.
d. Other: We must use a living animal from which to harvest live embryos and in which to
implant live embryos to produce live animals.
2.
THIS SPECIES HAS BEEN SELECTED BECAUSE:
a. A large database exists, allowing comparisons with previous data.
b. The anatomy or physiology is uniquely suited to the study because: rapid generation time;
desirable embryo grafts
c. This is, phylogentically, the lowest species that provides adequate size, tissue, or anatomy for
the proposed study.
d. It provides a particularly good model for duplicating the human condition.
e. Previous studies using this species formed the background of this project.
f.
It has the following unique features that make it the best available choice:
g. Other:
3.
POTENTIAL CONTRIBUTION(S) TO BIOLOGY AND/OR HUMAN MEDICINE:
These procedures may help produce more rapid and accurate accumulations of experimental results which
may benefit development of new medicines and may contribute to elucidation of the biochemical processes of
living organisms.
SECTION H - PAIN OR DISTRESS
1.
ANIMALS IN THIS STUDY HAVE THE FOLLOWING MANIPULATIONS (Select the appropriate option):
b)
Survival procedures, treatments or studies that could potentially cause pain or distress will be conducted.
SECTION I - ALTERNATIVES TO POTENTIALLY PAINFUL OR DISTRESSING PROCEDURES
1.
Please detail the specific procedures in this protocol which have the potential to cause pain or distress
(assume that any procedure which causes pain in a human has the potential to cause pain/distress in an animal)
Survival surgery to implant embryos in pseudopregnant mice.
2.
METHODS USED TO DETERMINE THAT ALTERNATIVES TO POTENTIALLY PAINFUL OR DISTRESSING
PROCEDURES WERE NOT AVAILABLE:
a. Data base or sources consulted (check one or more of the following):
JOHNS HOPKINS CENTER FOR ALTERNATIVES TO ANIMAL TESTING http://caat.jhsph.edu/
MEDLINE
BIOSIS
TOXLINE
Index Medicus
AGRICOLA
Biol. Abstracts
Animal Welfare Information Center
http://awic.nal.usda.gov/nal_display/index.php?info_center=3&tax_level=1&tax_subject=183
Current Research Information Service
Other Search Engine (describe): PubMed
1) Date of data base search: 08/25/10
2) Years covered by search: 1985-2010
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 9 of 13
3) Key words (2-3) used in search: rederivation, mouse laparotomy, mouse embryo
b. Scientific Meeting (name):
1) Date:
2) Discussion relevant to alternatives:
c. Scientific Journal (name):
1) Reference:
2) Discussion relevant to alternatives:
d. Consultation with Expert (name):
1) Qualifications of expert:
2) Date of consultation:
3) Discussion relevant to alternatives:
e. Provide a narrative of the methods or logic used in determining that alternatives to actually or potentially
distressful or painful procedures are not available. Describe any alternative to painful/distressful
procedures found in the literature. If a bona fide alternative was identified but not used, the
narrative should explain why:
http://altweb.jhsph.edu/searchalt.htm; http://www.aphis.usda.gov/ac/policy/policy12.pdf ;
http://www.nc3rs.org.uk/
Embryo transfer is a very successful method for efficient generation of mice where typical breeding schemes have
been unsuccessful for a variety of causes: low libido, advanced age of the animals, genetic background, or strain.
Live animals must be used: 1) to obtain viable embryos 2) as recipients of those embryos following collection to
produce viable offspring. This method saves time, per diem costs and the total number of animals used to achieve
the same results from regular breeding. In cases of severe pathogenic infections, this technique may be able to
recover the line, saving the cost in time, money, research and animals that otherwise might be wasted destroying an
entire mouse colony.
3.
EXPLAIN WHY DISTRESS OR PAIN CANNOT BE RELIEVED BY DRUGS
Anesthetics and analgesics are used to relieve pain or distress.
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 10 of 13
SECTION J - REGULATORY ASSURANCES
Signature Page
PI:
PROTOCOL TITLE: MOUSE EMBRYO OR SPERM CRYOPRESERVATION
1. ASSURANCE FROM THE PI OR COURSE DIRECTOR:
A. ASSURANCE FROM THE PI OR COURSE DIRECTOR
(check all that apply)
A. ALTERNATIVES TO PAINFUL PROCEDURES ASSURANCE: I certify that I have
considered alternatives to potentially painful/distressful procedures, as indicated in section H 2.
(check only if sectionH2 was required)
B. NON-DUPLICATIVE ASSURANCE: In planning this experiment, I have reviewed the relevant
literature (e.g., by a computer database literature search, use of comprehensive review articles,
or consultation with the Animal Welfare Information Center, etc.). Based on the available
literature, I certify that the activities involving animals described in this protocol do not
unnecessarily duplicate previous research. (required for research protocols)
C. RESEARCH STUDIES: I certify that the above statements are true. If this protocol is
associated with a grant application, I certify that this protocol is essentially the same as the study
found in grant application or program/project (see Section A for funding information). The
IACUC will be notified of any changes in the proposed project, or personnel, relative to this
application, prior to proceeding with any animal experimentation.
I will not proceed with animal experimentation until approval by the IACUC is granted. (required for
research protocols)
D. TEACHING EXERCISES: I certify that the information in this application is essentially the
same as contained in the course outline. The IACUC will be notified of any changes in the
proposed teaching exercises, or personnel, relative to this application, prior to proceeding with
any animal manipulation. I will not proceed with any animal manipulation until approval by the
IACUC is granted. (required for teaching protocols)
Principal Investigator
Date
2. VETERINARY REVIEW AND ASSURANCE
UC Denver Veterinarian
Date
3. IACUC CHAIR REVIEW AND ASSURANCE
UC Denver Chair
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
Date
(Revised11//10)
Page 11 of 13
APPENDIX A - SURVIVAL SURGICAL PROCEDURES
PI:
PROTOCOL TITLE: REDERIVATION OF MOUSE COLONIES IMPLANTATION OF CYROPRESERVED OR
FRESH EMBRYOS
Are animals expected to survive surgery and regain consciousness?
NO (If NO, delete Appendix A from your application as it is not applicable)
YES (If YES, complete the remainder of Appendix A)
1.
2.
3.
4.
5.
Species: Mouse
Surgeon's name: Abby Zamora; Saiphone Webb
Anesthetist's name: Abby Zamora; Saiphone Webb
Location where surgery will be done: P15 0384
Describe the pre-op preparation of the animals.
a. Food restricted for hours
b. Water restricted for hours
c. Pre-op medications given:
Please describe other preparations in detail.
6. Minimal sterile techniques will include (check all that will be used):
a. Sterile instruments
b. Sterile gloves for surgeon
c. Scrubs or laboratory coat (rodents only)
d. Sterile surgical gown (not required for rodents or aquatics)
e. Surgical mask and surgical cap
f. Sterile operating area - table, drapes
7.
Clipping of hair
h. Skin preparation with a sterilant such as betadine
i. Practices to maintain sterility of instruments during surgery
8. Describe the following surgical procedures:
j.
Incision size and location: ~0.5 cm incision. On flank ~ 1 cm caudal to rib cage and ~1.5 cm
lateral to spine
k.
Method of closure: body wall: 5.0 absorbable suture (Vicryl), skin: wound clip
l.
General surgical details: The pseudopregnant (PsPg) female is anesthetized using isoflurane
anesthetic using a veterinary vaporizer and given an injection of an analgesic (Banamine). Upon full
anesthesia (lack of toe/foot pinch response – 3-5 minutes), the mouse is placed on her right side under a
dissection microscope. The incision site is located approximately 1 cm caudal to the rib cage and 1.5 cm
lateral to the spine. The skin of the prepared surgical site is grasped with small forceps and a small
incision (0.5 cm) is made in the skin. The incision is moved around over the underlying body wall until the
ovarian fat pad beneath can be seen and a small incision is made at that point, taking care to avoid cutting
the vascular and nerve bundles in the muscle. The ovarian fat pad is grasped with forceps and gently
pulled through the incision, bringing with it the ovary, oviducts and proximal portion of the uterus.
Exteriorization of the reproductive tract is kept to the minimum necessary. A small bulldog clamp is
attached to the fat pad if necessary to keep the oviduct exteriorized. A small opening is blunt dissected in
the bursal membrane, allowing access to the ostium of the oviduct.
Twenty five to thirty washed embryos are loaded into a transfer pipet in sterile media. The pipet is
introduced through the hole in the bursa and threaded into the ostium of the oviduct. The embryos are
gently expelled into the ampullary end of the oviduct.
Alternately, a small puncture using a 27-30 ga. needle is made in the oviduct just distal to the ampulla.
The loaded embryo transfer pipet is inserted into the oviduct and the embryos expelled.
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 12 of 13
Following successful transfer, the mouse's reproductive organs are gently placed back into the body
cavity. A single 5.0 Vicryl (or other absorbable) suture is used to close the opening in the body wall if
necessary. The edges of the skin at the incision are opposed and closed with a wound clip. The wound
clip will be removed in <14 days.
The mouse is placed back into her cage to recover. Gentle heat may applied by placing one end of the
cage on a heating pad (aquamat) at low setting. Following recovery of the female in about 3-5 minutes,
she is transferred to the mouse room. Banamine will be administered 48-72 hours from the initial injection
m. Provisions for Post Surgical Care of the Animals:
a. Who will be responsible for post operative care? Abby Zamora; Saiphone Webb, Peter Koch
b. Post-operative analgesics be given: Banamine, 12 hr post op, if necessary
(1) Routinely to all animals: immediately prior to surgery and ~12 hr later if necessary.
(2) As needed, determined by:
c. Where and how will animals be recovered from anesthesia? Room P15 0384; animal will be
placed on a warming blanket (aquamat) to minimize heat loss if necessary.
d. How frequently will animals be monitored during anesthesia recovery? Every 3-5 minutes
e. What long term post-surgical care will be provided?
(1) Wound monitoring and/or care: Pain, discomfort, signs of infection, changes in behavior,
posture or activity, body weight
(2) Provision of analgesics: Banamine
(3) Fluid supplementation
(4) Special diet provisions
(5) Antibiotics (specify type, dose and route)
Please describe this care in detail.
n. Will any animal have more than one major survival surgical procedure?
NO
YES. If yes, then:
a. Time interval between surgeries:
b. Scientific necessity or rationale for performing more than one procedure in the same
animal instead of using more animals:
IACUC Application Form – Rederivation of Mouse Colonies: Implantation of Cryopreserved or Fresh Embryos
(Revised11//10)
Page 13 of 13