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					                       UNIVERSITY OF COLORADO DENVER
 STANDARDIZED PROTOCOL FOR PRODUCTION OF TRANSGENIC MICE BY DNA OR STEM CELL
                        INJECTION INTO MOUSE EMBRYOS

Note: Complete this protocol form only if you are having transgenic mice produced for you by the UCDenver Transgenic and Gene Targeting Core (TGTC).
Any breeding or manipulations you plan to perform on the transgenic mice produced under this protocol must be described on a separate experimental
protocol 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:                                                MAIL BOX:                         EMAIL:
     c.   PROTOCOL AFFILIATIONS:                         UC Denver           Barbara Davis
                                                                             Cancer Center                   Denver Health
2) CAMPUS/FACILITY WHERE RESEARCH IS CONDUCTED:
              ANSCHUTZ MEDICAL CAMPUS                              OTHER:
     a.    Location of Laboratory
          BUILDING P15 ROOM NUMBER 0382 (if using Transgenic Core)
     b. Location Of Area Where Animal Procedures Are Performed
          BUILDING P15 ROOM NUMBER 0382 (if using Transgenic Core)
3) PROTOCOL TITLE: Production of Transgenic Mice by DNA or Stem Cell Injection into Mouse Embryos
          TYPE OF PROTOCOL:                   NEW                  PILOT                REPLACEMENT FOR #:
4) PROTOCOL ASSOCIATES
     a. INVOLVED IN ANIMAL PROCEDURES: Joe Anderson; Abby Knight, Saiphone Webb (PQ forms on file)
     b. INVESTIGATOR LAB CONTACT PERSON:
                     TELEPHONE:                       MAIL BOX:                     EMAIL:
5) TOTAL NUMBER OF EACH SPECIES IN APPLICATION: Mouse

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)                      MOU Attached
     b. GRANT NUMBER/ID (If known):                            OGC ROUTING NUMBER:
              IN REVIEW, DATE:                                     AWARDED, DATE:
     c.   SPEEDTYPE Number:
7) INCLUDED IN THE APPENDIX (select all that apply):
               DEPARTMENTAL APPROVAL LETTER
               PERSONNEL QUALIFICATION FORMS: (please complete and SIGN a PQF for PI)




IACUC Application Form – Production of Transgenic Mice                                                          Revised 10/10            Page1 of 12
      SECTION A2 – INDIVIDUAL INVESTIGATOR SPECIFIC PROJECT INFORMATION


1. What specific transgenic or embryonic stem cell lines will be established in these mice?
          (Please note: these transgenes or ES lines must be included in your experimental protocol. You must submit a protocol amendment
               to include these animal models if necessary.)
Transgene Name(s):                                             ES Cell Line Name(s):
1:                                                             1:
2:                                                             2:
3:                                                             3:
4:                                                             4:
5:                                                             5:

Remember to update total number of animals in section B5 a. and/or b. and in Section A5.

2. Are there any known deleterious health effects associated with these genetic manipulations?

        If so, provide details of the health effect and how they will be monitored and/or alleviated.

3. What is the potential benefit, of establishing these transgenic animals, to human medicine or biology?



4. Will the resulting transgenic mice be housed and/or used at UC Denver?

          No. Where (at what institution) will the transgenic mice be housed/used?
          Yes. What is the number of the protocol that describes the use of the transgenic mice?

5. At what age will tail tissue be sampled for genotyping?

     21 days            Other – Please Justify:

6. Please remember to complete Section J on page 10.


Note:     The TMCL cannot accept DNA constructs or transgenic stem cells for injection if any resulting
          animals would require housing in a facility at more secure than ABSL2 containment level. If
          hazardous material use is planned please contact the IACUC Office for further advice prior to
          submission. See “Terms of Service” document on the core website.

http://www.ucdenver.edu/academics/colleges/medicalschool/centers/StemCell/Transgenics/Pages/default.a
        spx




IACUC Application Form – Production of Transgenic Mice                                                 Revised 10/10         Page2 of 12
                                             SECTION B - PROJECT DESCRIPTION


1.    PROJECT GOALS IN NON-SCIENTIFIC (i.e. “LAY”) LANGUAGE

      The Goal of this study is to produce mice which carry artificially induced genetic material (genes or gene
      fragments) into the mouse genome. The incorporated DNA becomes part of the normal mouse genome and
      allows the study and manipulation of specific genes in a living organism. (Individual Investigators must
      provide specific information about their DNA construct in section A2)

2.    NARRATIVE DESCRIPTION OF ANIMAL PROCEDURES

      Part 1: Pronuclear DNA Injection (PNI).

      For each day of microinjection, 25-30 three-week old donor females are superovulated (SO) by hormone
      injections (Pregnant Mare Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (hCG)) to
      stimulate to ovaries to produce increased numbers of mouse embryos. The superovulated embryo donors
      are mated to fertile males from the Transgenic Core's standing breeder colony. The mated donors are
      sacrificed by CO2 narcosis followed by cervical dislocation the next morning. The cumulus masses (fertile
      oocytes and cumulus cells) are dissected from the oviducts and the adherent cumulus cells removed by using
      enzyme(s). The fertile one-cell embryos are collected, cleaned with media and the DNA construct injected.

      Following the microinjections, pseudopregnant (PsPg) females are transferred from the TGTC’s recipient
      female colony onto the investigator’s production protocol and the embryos are surgically transferred to the
      oviducts or uterus (ES injections) of the PsPg recipient females as described in appendix A.
      Following the surgeries, the recipient females will be housed in static mouse cages in the TGTC barrier suite
      until birth. The litter is generally weaned in approximately seven weeks after birth. All pups are eartagged at
      weaning. Biopsies are taken from the putative transgenic founder mice and given to the investigator to be
      screened for successful integration of the transgene. Recipient mothers are euthanized following weaning of
      the litters.

      Pseudopregnancy (PsPg) is stimulated by mating mature female mice with vasectomized males from the
      Transgenic Laboratory's standing colony. Those females who show evidence of mating (presence of a vaginal
      plug) the next morning are available as recipients for the embryo transfers. The non-plugged females and
      unused plugged females are placed back in the Transgenic and Gene Targeting Core recipient female colony
      and will be reused in 10-14 days.

      Following screening for the transgene by the investigator, the non-transgenic pups will be sacrificed by
      CO2 narcosis followed by cervical dislocation, and the positive pups transferred to the investigator's
      appropriate IACUC protocol for breeding and experimental use.

      Part 2: Biopsies:

      Tissue samples will be taken from the pups for analysis indicating successful integration of the transgene in
      the mouse genome. 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 is allowed without anesthesia or
      ligature. (A waiver may be granted for tail snips for genotyping in mice over 28 days old if no more
      than 1.5mm tissue is taken and if the line requires the extra days after the normal weaning age for
      development. The need for this should be detailed by the individual investigator, in consultation with
      the Core, when completing the application as it must have specific IACUC approval).
      Very little bleeding occurs as the tail tip is cartilaginous at this age. 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 taken from isofluorane-anaesthetized mice by one of two methods. For
      amounts of 0.25 ml, retro orbital blood sampling is used. For lesser amounts (0.1 ml), the lateral tail vein will
      be nicked and the blood collected.

IACUC Application Form – Production of Transgenic Mice                                       Revised 10/10       Page3 of 12
      Part 3: Embryonic Stem Cell Injection (ESI).

      Twenty five three week-old mice are superovulated and mated as described in the previous section (2.1).

      The embryo donors are sacrificed at 3.5 days (84 hours) after mating and the uteri dissected out. Blastocysts
      are collected from the uteri by flushing using embryo culture medium.

      Embryonic stem cell injections are performed by injecting the stem cells into the cavity (blastocoel) of the 3.5
      day blastocysts. Following injections, the embryos are transferred to the uteri of 2.5-day PsPg recipients (2.5
      days after mating). Postoperative care and recovery is monitored as described in the previous section for
      oviductal transfers.

      Tissue samples are generally not required to assess integration of the transgene in this procedure. Usually a
      "marker" such as coat color is used to indicate integration of the transgenic embryonic stem cell into the host
      blastocyst and the resulting mouse will be a chimera with two different hair colors. Directed mating is primarily
      used to assess germ line transmission of the transgene.

      Following weaning of the putative transgenic animals, they are transferred the investigator's IACUC
      experimental protocol. The foster mothers receive embryo transfer surgery one time only and euthanized by
      CO2 narcosis following weaning of their transgenic pups


3.    STUDY END-POINT FOR ANIMALS
      Animals will be:
                    Euthanized following completion of the experimental protocol
                    Transferred to an experimental protocol


4.    ANIMAL IDENTIFICATION:
        a.     Species: Mouse
        b.     Strain (for rodents): any
        c.     Sex: male/female
        d.     Age and/or weight range: 3-4 weeks; 6 weeks – 4 months


5.    EXPERIMENTAL GROUPS AND NUMBERS:

         a.        Table 5a

                   Microinjections:

Experimental/Control Group Number of Animals per               Number of Repetitions          Total number used
         Names                    Group                            (per group)              in group (sum per row)
  Embryo donors
                                                    25                    2                                50
  (pronuclear injection)

  PsPg females                                       6                    2                                12

                                                            TOTAL PER MICROINJECTION:                      62

          b.        Total Number of animals required for all microinjection:

                     62 per construct. X # of Constructs = Total # Animals Requested Please indicate the
               number of constructs to be used and adjust the total number of animals accordingly. Please also enter
               the total number of animals required in section A 5



IACUC Application Form – Production of Transgenic Mice                                     Revised 10/10        Page4 of 12
         Table 5b

          Embryonic Stem Cell Injections:

Experimental/Control Group Number of Animals per                  Number of Repetitions           Total number used
         Names                    Group                               (per group)               in group (sum per row)
  Embryo donors
                                                    25                       2                                 50
  (blastocyst injection)

  PsPg females                                       6                       2                                 12

                                                          TOTAL PER STEM CELL INJECTION:                       62

          b.        Total Number of animals required for stem cell injection:

           62 per stem cell line X # of ES cell Lines = Total # Animals Requested Please indicate the number
             of constructs to be used and adjust the total number of animals accordingly. Please also enter the
             total number of animals required in section A 5. Remember to consider both ES injections and DNA
             microinjections in calculating the totals for section A 5.



     c. How was the number of animals in each group determined?
               Microinjections:
               Experience has shown that up to 25 donors are required to produce usable numbers of fertile embryos;
               we would like to collect 250-350 one-cell embryos for microinjections. 50%-70% of the embryos
               generally survive the construct injection process, resulting in 150 to 245 viable embryos. Twenty five
               live embryos are surgically transplanted into pseudopregnant (PsPg) recipients. Therefore, the number
               of animals used represents the minimum number of animals required to generate an adequate and
               usable number of transgenic founder mice from microinjections.

               Embryonic Stem Cell Injections:

               Fewer embryos are collected at this stage from the uteri of the blastocyst donors. Approximately 50%
               of the embryos collected will be suitable for blastocyst injection however injection survival is high - 95-
               100%. We would expect to harvest 75 - 125 usable embryos from this number of blastocyst donors and
               can implant between eight and twelve embryos into the uteri of the blast recipients.
               Experience shows that these numbers will generate usable numbers of chimeric animals.


          d.        How was the number of repetitions for each group determined?
               The numbers/group represent the number of animals used per injection-day. The number of
               repetitions is the anticipated maximum number of injection-days per construct.


6. EXPERIMENTAL AGENTS:

                                                                             Frequency of Duration of Treatment
    Experimental Agents                   Dose           Route)   Volume
                                                                                Dosing            Effect
  PMSG (Embryo donors)
                                         2.5-5 IU          IP      0.1 ml        Single Dose              48 hours

  hCG (Embryo donors)
                                         2.5-5 IU          IP      0.1 ml        Single Dose              12 hours




IACUC Application Form – Production of Transgenic Mice                                         Revised 10/10         Page5 of 12
7. NEUROMUSCULAR BLOCKING AGENTS (I.E., PARALYTICS): N/A


8. HAZARDOUS MATERIAL USE: Yes: PMSG and hCG




                                          SECTION C - ANESTHESIA AND ANALGESIA

     1.   PRE-ANESTHETIC/ANALGESIC OR SEDATIVE DRUGS: N/A
      DRUG           DOSE      ROUTE      VOLUME      FREQUENCY OF                                       DURATION OF
                                                            DOSING                                        TREATMENT
 Banamine          2.5 mg/kg   IP or SQ     50 ul    1 time Immediately prior                               12 hours
                                                            to surgery
   2.

2.    ANESTHETIC DRUGS :

          Drug                 Initial Dose         Supplemental         Route          For Which                Expected
                                                       Dose                             Procedure?              Time Under
                                                                                                                Anesthesia
                                    3%                   1.5-3%         Inhalation
       Isoflurane                                                                     Embryo Transfer           20 minutes
                                                                                      Laparotomies

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                        DOSE             ROUTE      FREQUENCY          DURATION         WHICH ANIMALS?
                                                                     OF DOSING            OF
                                                                                      TREATMENT
 Banamine                             2.5 mg/kg          IP or SQ   Once - 12 hours     12 hours           Recipient Females
                                                                     after surgery




                                            SECTION D - POTENTIAL HEALTH CHANGES

1. POTENTIAL HEALTH CHANGES
      a. Decreased food and water intake.                                             j.   Coma.
      b. Abscesses.                                                                   k.   Labored breathing.
      c. Dehydration.                                                                 l.   Hypothermia.
      d. Infection                                                                    m.   Hyperthermia.
      e. Malnutrition.                                                                n.   Skin abnormalities.
      f.  General weakness.                                                           o.   Paralysis.
      g. Diarrhea.                                                                    p.   Ataxia.
      h. Constipation or ileus.                                                       q.   Urinary incontinence.
      i.  Seizures.                                                                   r.   Excessive urination.
             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:
IACUC Application Form – Production of Transgenic Mice                                          Revised 10/10         Page6 of 12
                    x. None

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)
         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:
   d. If yes, justify the special conditions:
   e.

3. METHOD OF EUTHANASIA (check all that apply):
       f.  Carbon dioxide exposure (followed by cervical dislocation).




IACUC Application Form – Production of Transgenic Mice                             Revised 10/10     Page7 of 12
                            SECTION F - ANIMAL ORDERING AND HOUSING INFORMATION

1.    SOURCE (check one):
          a. Purchased from an approved vendor.

2.    ANIMAL HOUSING (Check all that apply):
          b. UC Denver Center for Comparative Medicine

3.    WILL ANIMALS BE REMOVED FROM THE ANIMAL HOUSING FACILITY?
          The IACUC strongly encourages researchers to utilize the animal procedural areas in the approved housing facilities. If this is not possible,
          time animals spend out of an approved facility, contact CCM animal facilities director for guidelines and requirements.
              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: There is no other way to obtain living embryos for transgenic integration


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;
               short estrus cycle, modest housing requirements (space, feed etc.).
           c. This is, phylogenetically, 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: Ability to respond to
               superovulation hormones to produce high numbers of fertile one-celled or blastocyst stage embryos
               required for transgenic injections and successful animal production.
           g. Other:




IACUC Application Form – Production of Transgenic Mice                                                                 Revised 10/10            Page8 of 12
                                               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 into 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):
1) Date of data base search: 7/30/2010
2) Years covered by search: 1970-2010
3) Key words (2-3) used in search: laparotomy, transgenic mouse, alternative
    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/


There are no means of producing transgenic or gene knockout/knockin mice without performing embryo transfer
using surgical procedures. A living recipient female mouse must be used to carry the embryos to term. No
alternative methods were discovered via Medline search.

3.        EXPLAIN WHY DISTRESS OR PAIN CANNOT BE RELIEVED BY DRUGS
          (skip this section unless Section H1c or H1d has been checked)

          Banamine: 2.5 mg/kg @ 12 hr intervals




IACUC Application Form – Production of Transgenic Mice                                            Revised 10/10        Page9 of 12
Office Use Only, Protocol #

                                         SECTION J - REGULATORY ASSURANCES
                                                       Signature Page

PI:
PROTOCOL TITLE:

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 (please state funding agency, project title & award ID and
        date of award)        . 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                                   Date




IACUC Application Form – Production of Transgenic Mice Revised 10/07                                         Page 10 of 12
Office Use Only, Protocol #

                            APPENDIX A - SURVIVAL SURGICAL PROCEDURES
PI:
PROTOCOL TITLE: Production of Transgenic Mice by DNA or Stem Cell Injection into Mouse 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.      Specie: Mouse
 2.      Surgeon's name: Joe Anderson, Saiphone Webb, Abby Knight
 3.      Anesthetist's name: Joe Anderson, Saiphone Webb, Abby Knight
 4.      Location where surgery will be done: P15, room 0382
 5.      Describe the pre-op preparation of the animals.
            a. Food restricted for         hours
            b. Water restricted for         hours
            c. Pre-op medications given for rederivation: Banamine
         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
            g. Clipping of hair
            h. Skin preparation with a sterilant such as betadine
            i. Practices to maintain sterility of instruments during surgery. Rinse in saline, hot bead
                sterilization
 7.
  Describe the following surgical procedures:
           a. Incision size and location: 0.5-1.0 cm incision on flank; location is ~ 1 – 1.5 cm caudal to rib
               cage and ~1 cm lateral to spine
           b. Method of skin closure: Wound clip
           c. General surgical details: The pseudopregnant (PsPg) female is given a pre-op SC injection of
               analgesic (Banamine: 2.5 mg/Kg) and is anesthetized by isofluorane inhalation. (The TGTC has
               the appropriate veterinarian vaporizer with full scavenging of the waste gasses.) 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, ampulla and distal 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.

                   Oviductal transfers (pronuclear injection): A small opening is blunt dissected in the bursal
                   membrane, allowing access to the ostium of the oviduct. Fifteen to twenty washed embryos are
                   loaded into a transfer pipet in sterile media. The pipet is introduced into the ostium of the oviduct
                   and the embryos 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.

                   Blastocyst injections (ES injection): The preparation and incision site are as described above.
                   The one uterine horn is grasped with small tweezers and pulled from the body cavity to straighten
                   the uterine horn. Using a 27-30 ga. needle, a small puncture is made in the uterine wall just distal
                   to the junction with the oviduct. The transfer pipet is loaded with 10-14 injected blastocysts and
                   carefully inserted deep into the uterus through the puncture and the embryos expelled.

IACUC Application Form – Production of Transgenic Mice Revised 10/07                                            Page 11 of 12
Office Use Only, Protocol #
                 Following successful transfer, the mouse's reproductive organs are gently placed back into the
                 body cavity. A single 5.0 vicryl absorbable suture loop is used to close the incision in the body
                 wall. 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 if still present (the female usually will remove it by herself.).
                 The mouse is placed back into her cage to recover (3-5 minutes) and returned to the mouse room
                 within a half hour.
                  If the animal is exhibiting sign of pain (hunching, stiffness) the next morning, another injection of
                 Banamine will be administered. No further analgesia is typically required.


 7.      Provisions for Post Surgical Care of the Animals:
             a. Who will be responsible for post operative care? Joe Anderson, Abby Knight, Saiphone Webb,
             b. Post-operative analgesics be given:
            (1) Routinely to all animals
            (2) As needed, determined by:
             c. Where and how will animals be recovered from anesthesia? Room P15-0382; animals
                 returned to their cage. Recovery from Isofluorane anesthesia takes place within 3-5 minutes.
             d. How frequently will animals be monitored during anesthesia recovery? Every 2-3 minutes
             e. What long term post-surgical care will be provided?
            (1) Wound monitoring and/or care
            (2) Provision of analgesics
            (3) Fluid supplementation
            (4) Special diet provisions
            (5) Antibiotics (specify type, dose and route)
         Please describe this care in detail. Wound monitoring
 8.      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 – Production of Transgenic Mice Revised 10/07                                          Page 12 of 12

				
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