LONDON REGIONAL TRANSGENIC AND GENE TARGETING FACILITY

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1 LONDON REGIONAL TRANSGENIC AND GENE TARGETING FACILITY SPERM CRYOPRESERVATION AND IN VITRO FERTILIZATION RESOURCE PACKAGE For information, contact: Dr. Christopher Pin, PhD, Scientific Director London Regional Transgenic and Gene Targeting Facility Phone: ext Ms. Linsay Drysdale, B.Sc., ES Cell Technician London Regional Transgenic and Gene Targeting Facility Phone: ext I. OUTLINE OF SERVICES The London Regional Transgenic and Gene Targeting (LRTGT) Facility provides services for investigators to preserve and bank genetically modified mouse lines through sperm cryopreservation and then rederive these and other lines via in vitro fertilization. Sperm cryopreservation is a simple, efficient and inexpensive method for archiving large numbers of genetically-engineered mice. It involves the isolation of mature sperm from the caudae epididymides of two fertile males followed by rapid freezing and storage of the sperm in liquid nitrogen. In vitro fertilization can reconstitute a mouse line from fresh or frozen sperm. It can also be used for embryo cryopreservation, to rapidly expand a mouse line from a sperm harvested from a single male, rescue lines where males are poor breeders and to rederive a mouse line into a Specific Pathogen Free (SPF) barrier facility. The standard process of in vitro fertilization involves mixing an aliquot of fresh or frozen sperm with oocytes isolated from superovulated females of the appropriate background. After 4 to 6 hours the oocytes are washed and incubated overnight. Following overnight incubation, all resulting 2-cell embryos are transferred to pseudopregnant females.

2 II. SERVICE DESCRIPTION Sperm Cryopreservation We will cryopreserve the sperm of two males from a given mouse line. For best results, we recommend that the client supply two breeding males between 3 and 6 months of age that have been mated 5 to 7 days prior to the cryopreservation session. Sperm cryopreservation yields 10 straws of frozen sperm per male. An extra straw from each cryopreservation will be frozen and used to perform an IVF to demonstrate the ability of the thawed sperm to produce fertilized embryos. Our sperm cryopreservation service includes: Harvesting and freezing sperm from two males per strain Quality control test consisting of thawing one straw and performing an IVF to demonstrate the viability of the cryopreserved sperm Long-term storage in liquid nitrogen Provide a written report detailing the specifics of the session Current modifications to the sperm cryopreservation method have greatly improved the quality of thawed sperm, particularly for C57BL/6 strains. However, even with these improvements, the fertilization capacity of thawed sperm can differ significantly among mouse lines and the fertility of the individual males used as sperm donors. In Vitro Fertilization We provide in vitro fertilization with fresh or frozen sperm based on the needs of the investigator. For best results, we recommend that the client supply two breeding males between 3 and 6 months of age that have been mated 5 to 7 days prior to the IVF session. If frozen sperm is used in the procedure we can use an aliquot of sperm cryopreserved by the LRTGT and stored on site or sperm that has been cryopreserved by an external institution and shipped directly to us. Our in vitro fertilization service includes: Collection of sperm from donor male mice or thawing of cryopreserved sperm Incubation of sperm with oocytes collected from superovulated females Transfer of resulting 2-cell embryos to pseudopregnant females

3 III. PRIOR TO SUBMITTING A SPERM CRYOPRESERVATION OR IVF REQUEST 1. Consult the Director or LRTGT Technician The LRTGT will work with the investigator to determine the timing of the request and provide feedback on the best options for preserving the line. In some cases, sperm cryopreservation may not be the best option. 2. Have a valid animal protocol that includes the cryopreservation or IVF protocol. We are working to establishing a blanket protocol within the LRTGT that covers cryopreservation and IVF, but do not currently have this available. The LRTGT technician can provide details to include in your protocol. 3. Ensure sufficient numbers of proven breeder males. The LRTGT recommends that two proven breeders between 3 and 6 months of age are supplied by the client. IV. SUBMITTING A SPERM CRYOPRESERVATION REQUEST 1. Complete sperm cryopreservation request form (see below). Submit a request form (see included form) to the LRTGT Director or technician, including an animal protocol number covering handling of the mice. We will work with you to establish a time frame that fits both the LRTGT and the investigator. 2. Set aside two proven males for the cryopreservation session. The client should set aside two breeding males between the ages of 3 and 6 months. Males should be mated a week prior to the cryopreservation and then rested for 5 to 7 days. V. ONCE WE HAVE A SPERM CRYOPRESERVATION REQUEST: The LRTGT staff will: 1. Collect fresh sperm from the caudal epididymides of the two male mice provided by the client. 2. Freeze 10 straws of sperm per male and store in liquid nitrogen. 3. Perform an in vitro fertilization using one straw of frozen sperm to determine its capacity to produce fertilized embryos. 4. Provide a written report detailing the specifics of the session. We will inform the client of the quality of the sperm at the time of collection and the results of the test thaw and IVF. The LRTGT maintains an electronic database of all genetically modified mouse lines frozen down. This database can include an in depth description of the frozen line with relevant publications, MTA forms, etc. Please inform the LRTGT Director if you do not wish to have this information available to other London-based researchers. NOTE: The fertilization capacity of thawed sperm can differ significantly among mouse lines

4 and the fertility of the individual males used as sperm donors. V. SUBMITTING AN IN VITRO FERTILIZATION REQUEST 1. Complete an in vitro fertilization request form (see below). Submit a request form (see included form) to the LRTGT Director or Technician, including an animal protocol number covering handling of the mice. We will work with you to establish a time frame that fits both the LRTGT and the investigator. 2. Set aside two proven males for the IVF session. The client should set aside two breeding males between the ages of 3 and 6 months. Males should be mated a week prior to the IVF and then rested for 5 to 7 days. The females used in the procedure will be provided or ordered by the LRTGT. -OR- 3. Provide the LRTGT with two aliquots of cryopreserved sperm. The LRTGT will thaw two straws of sperm that have been frozen by us and stored in our facility. Alternatively, two aliquots of sperm frozen by an external source may be used. These samples must be shipped directly to the LRTGT. All females used in the procedure will be ordered by the LRTGT. VI. ONCE WE HAVE THE IVF REQUEST: The LRTGT staff will: 1. Collect fresh sperm from the caudal epididymides of the male mice provided by the client. An aliquot of this sperm will be incubated for 4-6 hours with oocytes collected from superovulated females. If frozen sperm is used, the sperm will be thawed and allowed to capacitate prior to incubation with the oocytes. After incubation with the sperm, the oocytes will be washed and incubated overnight. 2. All resulting two-cell embryos will be transferred to pseudopregnant females. 3. Provide a written report detailing the specifics of the session. We will inform the client of the quality of the sperm collected or thawed, the number of fertilized embryos transferred, and the number of pups born. NOTE: In vitro fertilization results will vary with genetic background and the quality of the males used. We do not guarantee that any given IVF procedure will produce a large number of pups. If you need to change or cancel your session, you must inform the LRTGT at least 3 days in advance to avoid the minimum charge. VI. COSTS: Sperm cryopreservation $500 In vitro fertilization session $1000

5 IVF session cancellation without notification $50 Cost of hormones Cost of females Health Testing $500 (If required) Note: Additional costs may be incurred depending upon the strain of females used as oocyte donors.

6 VII. INFORMATION SPECIFIC FOR CRYOPRESERVATION/IVF: MOUSE LINE TO BE CRYOPRESERVED: UWO ANIMAL PROTOCOL NUMBER: HAS THIS LINE BEEN SUCCESSFULLY CRYOPRESERVED AND RECOVERED PREVIOUSLY: YES NO Male Female Strain / Background Genotype Homozygous/ Heterozygous Date of Birth or Age of Mice Total Number Available KNOWN PHENOTYPE OF THIS LINE: PRESENT LOCATION OF ANIMALS: PERFORM A TEST FREEZE AND THAW: YES NO IF NO TO TEST FREEZE AND THAW, A SIGNATURE IS REQUIRED I agree that The London Regional Transgenic and Gene Targeting Facility will use all reasonable efforts and will not be held responsible for poor results obtained without the prior knowledge of a test freeze and thaw. QUALITY CONTROL TEST PER SESSION (if able): YES NO INVESTIGATOR TO PERFORM THE FOLLOWING: Hormone Injections: YES NO TRAINING REQUIRED Check Plugs: YES NO TRAINING REQUIRED Oviduct Collection: YES NO TRAINING REQUIRED REPRODUCTIVE PERFORMANCE OF MOUSE LINE: Females responsive to superovulation: YES NO UNKNOWN Average litter size: Males reproductively challenged? If so please explain:

7 LONDON REGIONAL TRANSGENIC AND GENE TARGETING SERVICE REQUEST FORM: DATE: CONTACT INFORMATION: INVESTIGATOR NAME: DEPT. ROOM NO. BLDG. MAILING ADDRESS TEL FAX ADDRESS LAB CONTACT (IF DIFFERENT FROM PI) ADDRESS UWO ANIMAL PROTOCOL NUMBER (INTERNAL REQUESTS) SERVICE REQUESTED (please check all that apply): Pronuclear Injection Cryopreservation (are embryos heterozygous or homozygous?) Rederivation Sperm cryopreservation In vitro fertilization PROJECT DETAILS: TITLE: For Pronuclear Injection Requests, Transgene Vector Name: DNA Information: (concentration/volume/buffer/size (kb)): Short Description of Expectations (including potential developmental problems):

8 PLEASE ATTACH: 1) Gel photo of DNA sample for electroporation or pronuclear injection 2) Detailed plasmid map (including diagnostic restriction enzyme cut sites and fragment lengths) 3) Visual evidence that genotyping can be performed by PCR (Pronuclear Injection Request) 4) Information specific for cryopreservation of mouse embryos or sperm BILLING INFORMATION: Please complete the following information for billing purposes: NAME OF FINANCIAL OFFICER FINANCIAL OFFICER S TELEPHONE NUMBER CHARGE TO ACCOUNT NUMBER (INTERNAL REQUESTS) CHARGE TO PO NUMBER (EXTERNAL REQUESTS) PUROLATOR ACCOUNT NUMBER (FOR SHIPPING PURPOSES) PREFERRED MOUSE COURIER ACCOUNT NUMBER SIGNATURE OF ACCOUNT HOLDER This signature indicates your acceptance of all outlined charges in the LRTGT information packages. SUBMIT THIS FORM TO: DR. CHRISTOPHER PIN LONDON REGIONAL TRANSGENIC AND GENE TARGETING FACILITY VICTORIA RESEARCH LABS, ROOM A COMMISSIONERS ROAD E. LONDON, ON N6A 4L6 PHONE: (519) X53073 FAX: (519) Date Received: By:

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