Section: IACUC SOP#: (Ver )

Transcription

1 STANDARD OPERATING PROCEDURE PRINCETON UNIVERSITY Section: IACUC SOP#: (Ver ) TITLE: PRODUCTION OF GENETIC MUTANT MICE SCOPE: This SOP covers the production of genetic mutant mice using DNA microinjection 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. The treatment 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) are sacrificed by CO2 inhalation. The uterus is dissected using sterile technique and flushed with physiologic buffer to harvest eggs. c) Fertilized eggs (ie. those containing a male and female pronucleus) are injected with one picoliter of buffer containing the DNA construct of interest. (DNA constructs are composed of cdna s and genomic sequences originally cloned from animal and human sources). d) Injected fertilized eggs are incubated until reimplanted into pseudopregnant foster females. 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 and superovulate eggs. The treatment is single intraperitoneal (IP) injection of pregnant mares serum (PMSG) followed 48 hours later by a single IP injection of human chorionic gonadotropin (HcG). Females that have copulated (ie. contain vaginal plugs upon examination) are sacrificed by CO2 inhalation. The uterus is dissected using sterile technique and flushed with physiologic buffer to harvest blastocyst eggs. d) Blastocysts are further cultured before implantation into pseudopregnant foster females. 1

2 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 will diminish successful maintenance of the pregnancy. g) The incision is closed with wound clips and the mouse placed on a 35-37C warmed surface to recover from anesthesia. The mice are observed for recovery. Once able to maintain sternal recumbency and moving aroung, the mouse is moved to its home cage that contains ample soft bedding. h) Post-operative analgesics and antibiotics will not be used. Analgesia is not indicated since mice recover rapidly, show no overt signs of pain (ie. huddling, weight loss, anorexia, dehydration, teeth chattering, etc), may complicate anesthesia recovery with excessive sedation and hypothermia, and may complicate the successful establishment of pregnancy. Antibiotics are also not indicated since procedures are done using aseptic technique and incisions are open 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/or cautery. d) The incision is closed with wound clips and the mouse placed on a 35-37C warmed surface to recover from anesthesia. The mice are observed for recovery. Once able to maintain sternal recumbency and moving aroung, the mouse is moved to its home cage that contains ample soft bedding. e) Post-operative analgesics and antibiotics will not be used. Analgesia is not indicated since mice recover rapidly, show no overt signs of pain (ie. huddling, weight loss, anorexia, dehydration, teeth chattering, etc), may complicate anesthesia recovery with excessive sedation and hypothermia. Antibiotics are also not indicated since procedures are done using aseptic technique and incisions are open for a very short period of time. 2

3 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 pressure applied, 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 line status, 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 to obtain mice heterozygous for the mutation. Then, they are intercrossed to obtain homozygous mutant mice at a 1 in 4 ratio. 3

4 SEARCH OF THE RELEVANT LITERATURE FOR ALTERNATIVE PROCEDURES WHICH MAY CAUSE PAIN OR DISTRESS, BASED ON THE 3 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 this SOP. Databases: NLM Gateway: (3 Full Databases) ( AltWeb: ( Keywords: Cell culture OR computer model* OR invertebrate? OR nonanimal Alternative? AND (refinement OR replacement OR reduction) Mice AND (Transgenesis) Mice AND (Ovary Implantation) Mice AND (Superovulation) Mice AND PMSG Mice AND Avertin 4

5 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 surgical activity, 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 a clean, disinfected, and uncluttered area. Pre-operative care should include administration of sedatives and analgesics appropriate for the species and procedure. Conscious animals awaiting surgery should be guarded from sight, sound and smell of animals undergoing the procedure. Post-operative care is required and should consist of maintenance of body temperature, administration of analgesics (non-use must be justified below), and continuous monitoring until the animal is able to maintain sternal recumbency. The use of prophylactic antibiotics should be considered. Survival surgery has the potential to cause significant pain and distress when performed with inadequate anesthesia and analgesia. Please contact the Campus Veterinarian ( ) 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 USDA Animal Care Policy (4/14/97) states no animal is to be used in more than one major survival operative procedure except in cases of scientific necessity or veterinary care. If yes, provide complete scientific justification 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 been trained by senior laboratory personnel. 3. What is the approximate length of time required for surgical procedures? The surgical procedures take approximately 10 minutes to complete. 4. What pre-anesthetics, anesthetics, and other relevant drugs will be used? (Give agent, dose, routes of exposure, and frequency of administration) The anesthesia used in 2.5% Avertin at a dose of ml/g body weight administered I.P. 5. Will post-operative analgesics be used? Yes: No: If yes, give agent, dose, and frequency of administration. If no, explain fully. The objective of the surgeries is to impregnate the mice. Analgesics are not used so as not to interfere with the normal progression of pregnancy and embryonic development. 5

6 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 also would not routinely be given so as not to interfere with the normal progression of pregnancy and embryonic development. 7. To help the IACUC discuss this procedure, please describe any adverse effects that are expected or might result from this procedure. Comments: This procedure has become routine for transfer of embryo's to foster mothers. The procedure is required to rederive strains and produce transgenic mice. It is well documented that when properly done there is little risk to the mouse. 6

7 NLM Gateway Results Summary Mice AND Transgenesis Results Summary Category Items Found Journal Citations 164 Actions Books / Serials / AVs 1 Consumer Health 0 Meeting Abstracts 0 Other Collections 0 Total of 1 8/8/01 12:29 PM

8 NLM Gateway Results Mice AND Transgenesis Results Journal Citations ( 2 collections searched ) Displaying items 1-20 of 164 found Page 1 of 9 2 Pick A Collection 1 Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy? Wells T, Carter DA. J Neurosci Methods Jul 30;108(2): PMID: [PubMed - in process] From PubMed 2 [No title available] Grobet L. Bull Mem Acad R Med Belg. 2000;155(10-12):330-8; discussion French. PMID: [PubMed - in process] From PubMed 3 A specific promoter of the sensory cells of the inner ear defined by transgenesis. Boeda B, Weil D, Petit C. Hum Mol Genet Jul 15;10(15): PMID: [PubMed - in process] From PubMed 4 Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Ciana P, Di Luccio G, Belcredito S, Pollio G, Vegeto E, Tatangelo L, Tiveron C, Maggi A. Mol Endocrinol Jul;15(7): PMID: [PubMed - in process] From PubMed 5 Examining the In Vivo Role of the Amino Terminus of the Essential Myosin Light Chain. Sanbe A, Gulick J, Fewell J, Robbins J. J Biol Chem Jun 29; PMID: [PubMed - as supplied by publisher] From PubMed 1 of 4 8/8/01 12:12 PM

9 NLM Gateway Results Summary Mice AND "ovary implantation" Results Summary Category Items Found Journal Citations 257 Actions Books / Serials / AVs 0 Consumer Health 0 Meeting Abstracts 0 Other Collections 0 Total of 1 8/8/01 12:30 PM

10 NLM Gateway Results Summary Mice AND "superovulate" Results Summary Category Items Found Journal Citations 15 Actions Books / Serials / AVs 0 Consumer Health 0 Meeting Abstracts 0 Other Collections 0 Total 15 1 of 1 8/8/01 12:35 PM

11 NLM Gateway Results Summary Mice AND "pmsg" AND alternatives Results Summary Category Items Found Journal Citations 1 Actions Books / Serials / AVs 0 Consumer Health 0 Meeting Abstracts 0 Other Collections 0 Total 1 1 of 1 8/8/01 12:36 PM

12 NLM Gateway Results Mice AND "pmsg" AND alternatives Results Journal Citations ( 2 collections searched ) Displaying items 1-1 of 1 found Page 1 of 1 1 Pick A Collection 1 Laboratory methods of equine pregnancy diagnosis. Walker D. Vet Rec May 7;100(19): PMID: [PubMed - indexed for MEDLINE] From PubMed Journal Citations ( 2 collections searched ) Displaying items 1-1 of 1 found Page 1 of 1 1 Pick A Collection 1 of 1 8/8/01 12:36 PM

13 NLM Gateway Results Summary Mice AND Vasectomy Results Summary Category Items Found Journal Citations 59 Actions Books / Serials / AVs 7 Consumer Health 0 Meeting Abstracts 0 Other Collections 0 Total 66 1 of 1 8/8/01 12:38 PM

14 NLM Gateway Results Summary Mice AND Avertin Results Summary Category Items Found Journal Citations 16 Actions Books / Serials / AVs 0 Consumer Health 0 Meeting Abstracts 0 Other Collections 0 Total 16 1 of 1 8/8/01 12:39 PM

15 Altweb-Altweb Search Engine wysiwyg://218/ August 8, 2001 Alternatives A-Z HOME About Us Hot Topics Just for Kids World Congress CONTACT US home > Altweb Search Engine Acronyms Best Practices Calendar Databases Educational Resources Endocrine Disruptors FAQs Funding Glossary History HPV Testing Humane Endpoints IACUCS Journals Just for Kids Links MABs News Pain and Distress Project Teams Publications Rats, Mice and Birds Regulations Russell and Burch Search Engine Search Templates Search Tutorials Slides/Overheads Sponsors Statistics Support Us TestSmart Three Rs Transgenics World Congress Enter words or phrases you wish to search... Search Results Your search for "Avertin" matched 2 of documents. Score Title Table of Contents.htm Summary: The effects of housing, feeding time and diet composition on the behaviour of the laboratory rabbit were examined. BALBc mice given nesting material showed less weight gain and smaller brown adipose tissue weights than animals without nesting material. ANZCCART Fact Sheets: Experimental Techniques and Anaesthesia in the Rat and the Mouse Summary: However, progress of experiments and the quality of life of experimental animals is closely related to the ability and training of research personnel and animal care staff handling the animals. A number of mechanical devices can be used to restrain rats Copyright The Johns Hopkins Center For Alternatives To Animal Testing / altweb@jhsph.edu. All rights reserved. 1 of 2 8/8/01 12:41 PM

16 Altweb-Altweb Search Engine wysiwyg://221/ August 8, 2001 Alternatives A-Z HOME About Us Hot Topics Just for Kids World Congress CONTACT US home > Altweb Search Engine Acronyms Best Practices Calendar Databases Educational Resources Endocrine Disruptors FAQs Funding Glossary History HPV Testing Humane Endpoints IACUCS Journals Just for Kids Links MABs News Pain and Distress Project Teams Publications Rats, Mice and Birds Regulations Russell and Burch Search Engine Search Templates Search Tutorials Slides/Overheads Sponsors Statistics Support Us TestSmart Three Rs Transgenics World Congress Enter words or phrases you wish to search... Search Results Your search for "Mice AND Transgenesis" matched 3 of documents. Score Title Housing, Husbandry, and Welfare of Rodents, QB Summary: Clark, F.; Willan, P. C. Welfare and science proceedings of the Fifth Symposium of the Federation of European Laboratory Animal Science Associations, 8-11 June 1993, Brighton, UK / Federation of European Laboratory Animal Science Associations Symposium. Transgenic Animals Summary: TITLE: Transgenic Animals PUBLICATION DATE: June, 1994 ENTRY DATE: June, 1994 EXPIRATION DATE: None UPDATE FREQUENCY: As needed CONTACT: Biotechnology Information Center(biotech@nalusda.gov) National Agricultural Library DOCUMENT TYPE: Text DOCUMENT SIZE: News 13/2 Summary: Gene targeted animals, such as knockout mice, are created through the genetic manipulation of embryonic stem (ES) cells, which are then used to generate a new mouse strain. The role of genetically modified mice in science today Genetically modified mice Copyright The Johns Hopkins Center For Alternatives To Animal Testing / altweb@jhsph.edu. All rights reserved. 1 of 2 8/8/01 12:43 PM

17 Altweb-Altweb Search Engine wysiwyg://223/ August 8, 2001 Alternatives A-Z HOME About Us Hot Topics Just for Kids World Congress CONTACT US home > Altweb Search Engine Acronyms Best Practices Calendar Databases Educational Resources Endocrine Disruptors FAQs Funding Glossary History HPV Testing Humane Endpoints IACUCS Journals Just for Kids Links MABs News Pain and Distress Project Teams Publications Rats, Mice and Birds Regulations Russell and Burch Search Engine Search Templates Search Tutorials Slides/Overheads Sponsors Statistics Support Us TestSmart Three Rs Transgenics World Congress Enter words or phrases you wish to search... Search Results Your search for "Mice AND Superovulation" matched 13 of documents. Score Title 1 2 [Next] qb9323 Summary: 1. Livestock--Embryos--Transplantation--Bibliography. Application models of embryo transfer techniques in cattle. Science; 1992 Apr. Journal of animal science v. 70 (4): p. Transgenic Animals Summary: TITLE: Transgenic Animals PUBLICATION DATE: June, 1994 ENTRY DATE: June, 1994 EXPIRATION DATE: None UPDATE FREQUENCY: As needed CONTACT: Biotechnology Information Center(biotech@nalusda.gov) National Agricultural Library DOCUMENT TYPE: Text DOCUMENT SIZE: qb9410 Summary: Urge FDA to label milk from BGH-injected cows. Mar. Journal of dairy science v. 74 (3): p. production responses and associated changes in DM intake. 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 / American Association for Laboratory Animal Science ; 84-1). Includes references. Beyers, T.M. Laboratory Animal Facilities and Management, QB Summary: (Quick bibliography series ; 95-17) 1. Laboratory animals--management--bibliography. Besch, E.L.; Brigmon, R.L. Cordova, Tenn. : American Association for Laboratory Animal Science; 1991 Jan. Laboratory animal science v. 41 (1): 1 of 3 8/8/01 12:44 PM

18 Altweb-Altweb Search Engine wysiwyg://226/ August 8, 2001 Alternatives A-Z HOME About Us Hot Topics Just for Kids World Congress CONTACT US home > Altweb Search Engine Acronyms Best Practices Calendar Databases Educational Resources Endocrine Disruptors FAQs Funding Glossary History HPV Testing Humane Endpoints IACUCS Journals Just for Kids Links MABs News Pain and Distress Project Teams Publications Rats, Mice and Birds Regulations Russell and Burch Search Engine Search Templates Search Tutorials Slides/Overheads Sponsors Statistics Support Us TestSmart Three Rs Transgenics World Congress Enter words or phrases you wish to search... Search Results Your search for "Mice AND PMSG" matched 10 of documents. Score Title qb9323 Summary: 1. Livestock--Embryos--Transplantation--Bibliography. Application models of embryo transfer techniques in cattle. Science; 1992 Apr. Journal of animal science v. 70 (4): p. Transgenic Animals Summary: TITLE: Transgenic Animals PUBLICATION DATE: June, 1994 ENTRY DATE: June, 1994 EXPIRATION DATE: None UPDATE FREQUENCY: As needed CONTACT: Biotechnology Information National Agricultural Library DOCUMENT TYPE: Text DOCUMENT SIZE: qb9410 Summary: Urge FDA to label milk from BGH-injected cows. Mar. Journal of dairy science v. 74 (3): p. production responses and associated changes in DM intake. qb9436 Summary: Amsterdam : Elsevier Science Publishers B.V.; 1988 May. East Melbourne : Commonwealth Scientific and Industrial Research Organization; Abstract: Animal behavior has been defined as the interaction of an animal with its environment. Anesthesia and Analgesia for Companion and Laboratory Animals, QB Summary: Day, T.K.; Muir, W.W. III Schaumburg, Ill. : American Veterinary Medical Association; 1993 Jan. American journal 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 qb of 3 8/8/01 12:45 PM