standard operating procedure - princeton …s/sop-mutantmice.pdf · scope: this sop covers the...

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STANDARD♦♦♦♦ OPERATING♦♦♦♦ PROCEDURE

PRINCETON UNIVERSITY

Section: IACUC SOP#: (Ver.8-2001)

TITLE: PRODUCTION OF GENETIC MUTANT MICE

SCOPE: This SOP covers the production of genetic mutant mice using DNAmicroinjection technique and gene targeting in ES cells (KnockOut/KnockIn).

POLICY: The production of genetic mutants is a commonly used standard procedure.

PROCEDURES:

1) Generation of transgenic mice via pronuclear injection.a) Male and female mice are caged together for mating. In some instances, the

female will have been treated to synchronize estrus and superovulate eggs. Thetreatment is single intraperitoneal (IP) injection of pregnant mares serum (PMSG)followed 48 hours later by a single IP injection of human chorionic gonadotropin(HcG).

b) Females that have copulated (ie. contain vaginal plugs upon examination) aresacrificed by CO2 inhalation. The uterus is dissected using sterile technique andflushed with physiologic buffer to harvest eggs.

c) Fertilized eggs (ie. those containing a male and female pronucleus) are injectedwith one picoliter of buffer containing the DNA construct of interest. (DNAconstructs are composed of cDNA’s and genomic sequences originally clonedfrom animal and human sources).

d) Injected fertilized eggs are incubated until reimplanted into pseudopregnant fosterfemales.

2) Generation of mutant mice via gene targeting in ES cells.a) Specific mutations are introduced in cultured mouse embryonic stem (ES) cells.b) These ES cells are subsequently injected into 3 and one-half day old blastocyst

embryo’s.c) To generate blastocysts, male and female mice are caged together for mating. In

some instances, the female will have been treated to synchronize estrus andsuperovulate eggs. The treatment is single intraperitoneal (IP) injection ofpregnant mares serum (PMSG) followed 48 hours later by a single IP injection ofhuman chorionic gonadotropin (HcG). Females that have copulated (ie. containvaginal plugs upon examination) are sacrificed by CO2 inhalation. The uterus isdissected using sterile technique and flushed with physiologic buffer to harvestblastocyst eggs.

d) Blastocysts are further cultured before implantation into pseudopregnant fosterfemales.

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3) Manipulation of Pseudopregnant Foster Female Mice.a) Female mice are housed with previously vasectomized male mice for mating.b) Females that have copulated (ie. contain vaginal plugs upon examination, but no

sperm) are taken for uterine implantation of cultured oocytes.c) The mouse is anesthetized with AVERTIN (Tribromoethanol) anesthetic (125

mg/kg IP, prepared fresh daily).d) The fur is clipped and skin is swabbed with 70% alcohol to remove excess skin

oils and provide mild disinfection.e) Using sterile technique, an incision is in the inguinal region and the oviduct and

uterine horn exposed by gentle pulling.f) The cultured oocytes are injected into the oviduct ampulla or through the uterine

wall. Great care is taken not to cause laceration or bleeding since this willdiminish successful maintenance of the pregnancy.

g) The incision is closed with wound clips and the mouse placed on a 35-37Cwarmed surface to recover from anesthesia. The mice are observed for recovery.Once able to maintain sternal recumbency and moving aroung, the mouse ismoved to its home cage that contains ample soft bedding.

h) Post-operative analgesics and antibiotics will not be used. Analgesia is notindicated since mice recover rapidly, show no overt signs of pain (ie. huddling,weight loss, anorexia, dehydration, teeth chattering, etc), may complicateanesthesia recovery with excessive sedation and hypothermia, and maycomplicate the successful establishment of pregnancy. Antibiotics are also notindicated since procedures are done using aseptic technique and incisions areopen for a very short period of time.

4) Preparation of Vasectomized Male Mice.a) The mouse is anesthetized with AVERTIN (Tribromoethanol) anesthetic (125

mg/kg IP, prepared fresh daily).b) The fur is clipped and skin is swabbed with 70% alcohol to remove excess skin

oils and provide mild disinfection.c) Using sterile technique, an incision is in the inguinal region and the vas deferens

exposed by gentle pulling. The vas are individually obstructed by ligation and/orcautery.

d) The incision is closed with wound clips and the mouse placed on a 35-37Cwarmed surface to recover from anesthesia. The mice are observed for recovery.Once able to maintain sternal recumbency and moving aroung, the mouse ismoved to its home cage that contains ample soft bedding.

e) Post-operative analgesics and antibiotics will not be used. Analgesia is notindicated since mice recover rapidly, show no overt signs of pain (ie. huddling,weight loss, anorexia, dehydration, teeth chattering, etc), may complicateanesthesia recovery with excessive sedation and hypothermia. Antibiotics are alsonot indicated since procedures are done using aseptic technique and incisions areopen for a very short period of time.

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5) Manipulation of Mouse Pups.a) At the time of weaning, mouse pups are identified with a standard ear tag.b) A single, small piece of tail tip is removed for DNA analysis. The tip is cut with

sterile scalpel or razor blade. The tip is monitored for bleeding and direct pressureapplied, as needed. The tip is immediately frozen in liquid nitrogen or on dry ice.

c) Transgenic mouse pups via pronuclear injection are bred to ascertain germ linestatus, phenotypic analysis, and molecular studies.

d) Mutant mice via gene targeting in ES cells are bred towards homozygousity,pheotypic analysis, and molecular studies. Chimeras are crossed with wild-type toobtain mice heterozygous for the mutation. Then, they are intercrossed to obtainhomozygous mutant mice at a 1 in 4 ratio.

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SEARCH OF THE RELEVANT LITERATURE FOR ALTERNATIVEPROCEDURES WHICH MAY CAUSE PAIN OR DISTRESS, BASED ON THE3’Rs OF ANIMAL RESEARCH

General Statement:The literature search will be conducted annually for alternative procedures

and attached to this SOP. Each IACUC protocol will contain the written Narrative(see USDA Policy #12), however the database literature search will be within thisSOP.

Databases:NLM Gateway: (3 Full Databases)

(http://gateway.nlm.nih.gov/gw/Cmd)AltWeb: (http://www.altwebsearch.com/)

Keywords:Cell culture OR computer model* OR invertebrate? OR nonanimalAlternative? AND (refinement OR replacement OR reduction)Mice AND (Transgenesis)Mice AND (Ovary Implantation)Mice AND (Superovulation)Mice AND “PMSG”Mice AND “Avertin”

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PRINCETON IACUC SURVIVAL SURGERY ATTACHMENT

CASE #

Title: Generation of Mutant Mice and Behavioral Studies

Survival surgery in all species must be conducted utilizing aseptic techniques, in areas dedicated for surgicalactivity, and by personnel trained in the procedure. Aseptic technique requires, but should not be limited to,use of sterile instruments and surgical gloves, and surgeon mask. Sterile procedures must be conducted in aclean, disinfected, and uncluttered area.

Pre-operative care should include administration of sedatives and analgesics appropriate for the species andprocedure. Conscious animals awaiting surgery should be guarded from sight, sound and smell of animalsundergoing the procedure.

Post-operative care is required and should consist of maintenance of body temperature, administration ofanalgesics (non-use must be justified below), and continuous monitoring until the animal is able to maintainsternal recumbency. The use of prophylactic antibiotics should be considered.

Survival surgery has the potential to cause significant pain and distress when performed with inadequateanesthesia and analgesia. Please contact the Campus Veterinarian (258-6246) for help with dosages,procedures, and monitoring criteria.

1. Will more than one major operative procedure be used for a given animal? Yes: No: The USDAAnimal Care Policy (4/14/97) states “no animal is to be used in more than one major survival operativeprocedure except in cases of scientific necessity or veterinary care.” If yes, provide complete scientificjustification and include key references.

2. Have personnel involved received training in aseptic surgical technique? Yes: No: If yes, list training for each. Personnel performing surgical techniques have beentrained by senior laboratory personnel.

3. What is the approximate length of time required for surgical procedures? The surgical procedurestake approximately 10 minutes to complete.

4. What pre-anesthetics, anesthetics, and other relevant drugs will be used? (Give agent, dose, routes ofexposure, and frequency of administration) The anesthesia used in 2.5% Avertin at a doseof 0.015 ml/g body weight administered I.P.

5. Will post-operative analgesics be used? Yes: No: If yes, give agent, dose, and frequency ofadministration. If no, explain fully. The objective of the surgeries is to impregnate themice. Analgesics are not used so as not to interfere with the normal progression ofpregnancy and embryonic development.

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6. Will antibiotics be used? Yes: No If yes, give agent, dose, and frequency of administration. If no,explain fully. Aseptic technique results in antibiotics being unnecessary. They alsowould not routinely be given so as not to interfere with the normal progression ofpregnancy and embryonic development.

7. To help the IACUC discuss this procedure, please describe any adverse effects that are expected or mightresult from this procedure.

Comments:This procedure has become routine for transfer of embryo's to foster mothers. Theprocedure is required to rederive strains and produce transgenic mice. It is welldocumented that when properly done there is little risk to the mouse.

Results Summary

CategoryItemsFound

Actions

Journal Citations 164

Books / Serials / AVs 1

Consumer Health 0

Meeting Abstracts 0

Other Collections 0

Total 165

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Mice AND Transgenesis

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NLM Gateway Results Summary http://gateway.nlm.nih.gov/gw/Cmd?GMResultsSummary

Results

Journal Citations ( 2 collections searched ) Displaying items 1 - 20 of 164 found

Page 1 of 9

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Genetic engineering of neural function in transgenic rodents: towards acomprehensive strategy?Wells T, Carter DA.J Neurosci Methods. 2001 Jul 30;108(2):111-30. PMID: 11478971 [PubMed - in process]From PubMed

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[No title available]Grobet L.Bull Mem Acad R Med Belg. 2000;155(10-12):330-8; discussion338-40. French. PMID: 11475895 [PubMed - in process]From PubMed

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A specific promoter of the sensory cells of the inner ear defined bytransgenesis.Boeda B, Weil D, Petit C.Hum Mol Genet. 2001 Jul 15;10(15):1581-9. PMID: 11468276 [PubMed - in process]From PubMed

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Engineering of a mouse for the in vivo profiling of estrogen receptoractivity.Ciana P, Di Luccio G, Belcredito S, Pollio G, Vegeto E, Tatangelo L, Tiveron C, Maggi A.Mol Endocrinol. 2001 Jul;15(7):1104-13. PMID: 11435611 [PubMed - in process]From PubMed

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Examining the In Vivo Role of the Amino Terminus of the EssentialMyosin Light Chain.Sanbe A, Gulick J, Fewell J, Robbins J.J Biol Chem. 2001 Jun 29;PMID: 11432848 [PubMed - as supplied by publisher]From PubMed

Mice AND Transgenesis

2 Pick A Collection

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Results Summary

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Consumer Health 0

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Total 257

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Mice AND "ovary implantation"

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Consumer Health 0

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Total 15

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Mice AND "superovulate"

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Results Summary

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Journal Citations 1


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Other Collections 0

Total 1

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Mice AND "pmsg" AND alternatives

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Page 1 of 1

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Laboratory methods of equine pregnancy diagnosis.Walker D.Vet Rec. 1977 May 7;100(19):396-9. PMID: 405788 [PubMed - indexed for MEDLINE]From PubMed


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Mice AND "pmsg" AND alternatives

1 Pick A Collection

1 Pick A Collection

1 of 1 8/8/01 12:36 PM

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Consumer Health 0

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Total 66

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Mice AND Vasectomy

1 of 1 8/8/01 12:38 PM


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Consumer Health 0

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Total 16

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Mice AND Avertin

1 of 1 8/8/01 12:39 PM

NLM Gateway Results Summary http://gateway.nlm.nih.gov/gw/Cmd?GMBasicSearch

August 8, 2001

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Your search for "Avertin" matched 2 of 522774 documents.

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Table of Contents.htm Summary: The effects of housing, feeding time and dietcomposition on the behaviour of the laboratory rabbit wereexamined. BALBc mice given nesting material showed lessweight gain and smaller brown adipose tissue weights thananimals without nesting material.

ANZCCART Fact Sheets: Experimental Techniques andAnaesthesia in the Rat and the Mouse Summary: However, progress of experiments and the qualityof life of experimental animals is closely related to the abilityand training of research personnel and animal care staffhandling the animals. A number of mechanical devices can beused to restrain rats

©Copyright 1997-2001 The Johns Hopkins Center For Alternatives To Animal Testing / [email protected]. All rights reserved.

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Housing, Husbandry, and Welfare of Rodents, QB 97-04 Summary: Clark, F.; Willan, P. C. Welfare and scienceproceedings of the Fifth Symposium of the Federation ofEuropean Laboratory Animal Science Associations, 8-11 June1993, Brighton, UK / Federation of European Laboratory AnimalScience Associations Symposium.

Transgenic Animals Summary: TITLE: Transgenic Animals PUBLICATION DATE:June, 1994 ENTRY DATE: June, 1994 EXPIRATION DATE: NoneUPDATE FREQUENCY: As needed CONTACT: BiotechnologyInformation Center([email protected]) National AgriculturalLibrary DOCUMENT TYPE: Text DOCUMENT SIZE:

News 13/2 Summary: Gene targeted animals, such as knockout mice,are created through the genetic manipulation of embryonicstem (ES) cells, which are then used to generate a new mousestrain. The role of genetically modified mice in science todayGenetically modified mice

©Copyright 1997-2001 The Johns Hopkins Center For Alternatives To Animal Testing / [email protected]. All rights reserved.

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qb9323 Summary: 1.Livestock--Embryos--Transplantation--Bibliography. Applicationmodels of embryo transfer techniques in cattle. Science; 1992Apr. Journal of animal science v. 70 (4): p.


qb9410 Summary: Urge FDA to label milk from BGH-injected cows.Mar. Journal of dairy science v. 74 (3): p. production responsesand associated changes in DM intake.

http://netvet.wustl.edu/species/rabbits/qb9416.txt Summary: Besch, E.L.; Brigmon, R.L. Cordova, Tenn. :American Association for Laboratory Animal Science; 1991 Jan.Laboratory animal science v. 41 (1): p. (Publication / AmericanAssociation for Laboratory Animal Science ; 84-1). Includesreferences. Beyers, T.M.

Laboratory Animal Facilities and Management, QB 95-17 Summary: (Quick bibliography series ; 95-17) 1. Laboratoryanimals--Management--Bibliography. Besch, E.L.; Brigmon,R.L. Cordova, Tenn. : American Association for LaboratoryAnimal Science; 1991 Jan. Laboratory animal science v. 41 (1):

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qb9323 Summary: 1.Livestock--Embryos--Transplantation--Bibliography. Applicationmodels of embryo transfer techniques in cattle. Science; 1992Apr. Journal of animal science v. 70 (4): p.


qb9410 Summary: Urge FDA to label milk from BGH-injected cows.Mar. Journal of dairy science v. 74 (3): p. production responsesand associated changes in DM intake.

qb9436 Summary: Amsterdam : Elsevier Science Publishers B.V.;1988 May. East Melbourne : Commonwealth Scientific andIndustrial Research Organization; 1990. Abstract: Animalbehavior has been defined as the interaction of an animal withits environment.

Anesthesia and Analgesia for Companion and LaboratoryAnimals, QB 95-12 Summary: Day, T.K.; Muir, W.W. III Schaumburg, Ill. :American Veterinary Medical Association; 1993 Jan. Americanjournal of veterinary research v. 54 (1): p. Lemke, K.A.;Tranquilli, W.J.; Thurmon, J.C.; Benson, G.J.; Olson, W.A.Schaumburg, Ill. : American Vete

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