Sociocultural changes in the human–animal relationship have led to increasing demands for animal welfare in biomedical research. The 3R concept is the basis for bringing this demand into practice: Replace animal experiments with alternatives where possible, Reduce the number of animals used to a scientifically justified minimum and Refine the procedure to minimize animal harm. The generation of gene-modified sentient animals such as mice and rats involves many steps that include various forms of manipulation. So far, no coherent analysis of the application of the 3Rs to gene manipulation has been performed. Here we provide guidelines from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science to implement the 3Rs in every step during the generation of genetically modified animals. We provide recommendations for applying the 3Rs as well as success/intervention parameters for each step of the process, from experiment planning to choice of technology, harm–benefit analysis, husbandry conditions, management of genetically modified lines and actual procedures. We also discuss future challenges for animal welfare in the context of developing technologies. Taken together, we expect that our comprehensive analysis and our recommendations for the appropriate implementation of the 3Rs to technologies for genetic modifications of rodents will benefit scientists from a wide range of disciplines and will help to improve the welfare of a large number of laboratory animals worldwide.
This is a preview of subscription content, access via your institution
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Russel, W. M. S. & Burch, R. L. The principles of humane experimental technique. (Methuen, 1959).
Kaneko, T., Sakuma, T., Yamamoto, T. & Mashimo, T. Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci. Rep. 4, 6382 (2014).
Hashimoto, M. & Takemoto, T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci. Rep. 5, 11315 (2015).
Chen, S., Lee, B., Lee, A. Y., Modzelewski, A. J. & He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 291, 14457–14467 (2016).
Wang, W. et al. Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. J. Genet. Genomics 43, 319–327 (2016).
Tröder, S. E. et al. An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS ONE 13, e0196891 (2018).
Takahashi, G. et al. GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice. Sci. Rep. 5, 11406 (2015).
Kobayashi, Y. et al. Modification of i-GONAD suitable for production of genome-edited C57BL/6 inbred mouse strain. Cells 9, 957 (2020).
Sato, M. et al. Sequential i-GONAD: an improved in vivo technique for CRISPR/Cas9-based genetic manipulations in mice. Cells 9, 546 (2020).
Advanced Protocols for Animal Transgenesis: An ISTT Manual. (Springer-Verlag, 2011); https://doi.org/10.1007/978-3-642-20792-1
Behringer R., Gertsenstein, M. Manipulating the mouse embryo: a laboratory manual. (Cold Spring Harbor Laboratory Press, 2014).
Moltó, V.-G., Montoliu, L., Pease, S. & Saunders, T. in Advanced Protocols for Animal Transgenesis: An ISTT Manual (eds Pease, S. & Saunders, T. L.) 81–97 (Springer-Verlag, 2011).
Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A. & Ruddle, F. H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl Acad. Sci. USA 77, 7380–7384 (1980).
Brinster, R. L. et al. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223–231 (1981).
Costantini, F. & Lacy, E. Introduction of a rabbit β-globin gene into the mouse germ line. Nature 294, 92–94 (1981).
Fielder, T. J. in Advanced Protocols for Animal Transgenesis: An ISTT Manual (ed. Pease, S.) 81–97 (Springer-Verlag, 2011).
Lindner, L. et al. Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents. Methods 191, 107–119 (2021).
Henikoff, S. Conspiracy of silence among repeated transgenes. BioEssays 20, 532–535 (1998).
Goodwin, L. O. et al. Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res. 29, 494–505 (2019).
Chiang, C. et al. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat. Genet. 44, 390–397 (2012). S1.
Cain-Hom, C. et al. Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification. Nucleic Acids Res. 45, e62 (2017).
Mukai, H. Y. et al. Transgene insertion in proximity to the c-myb gene disrupts erythroid–megakaryocytic lineage bifurcation. Mol. Cell. Biol. 26, 7953–7965 (2006).
Durkin, M. E., Keck-Waggoner, C. L., Popescu, N. C. & Thorgeirsson, S. S. Integration of a c-myc transgene results in disruption of the mouse Gtf2ird1 gene, the homologue of the human GTF2IRD1 gene hemizygously deleted in Williams–Beuren syndrome. Genomics 73, 20–27 (2001).
de Vree, P. J. P. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat. Biotechnol. 32, 1019–1025 (2014).
Blondal, T. et al. Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing. Methods 191, 68–77 (2021).
Hart-Johnson, S. & Mankelow, K. Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals. Lab. Anim. https://doi.org/10.1177/00236772211007306 (2021).
Remy, S. et al. The use of lentiviral vectors to obtain transgenic rats. Methods Mol. Biol. 597, 109–125 (2010).
Giraldo, P. & Montoliu, L. Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 10, 83–103 (2001).
Van Keuren, M. L., Gavrilina, G. B., Filipiak, W. E., Zeidler, M. G. & Saunders, T. L. Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res. 18, 769–785 (2009).
Chandler, K. J. et al. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. Mamm. Genome 18, 693–708 (2007).
Dubose, A. J. et al. Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene. Nucleic Acids Res. 41, e70 (2013).
Rostovskaya, M. et al. Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes. Genes 51, 135–141 (2013).
Zhao, L., Ng, E. T. & Koopman, P. A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis. Dev. Dyn. 243, 1086–1094 (2014).
Suster, M. L., Sumiyama, K. & Kawakami, K. Transposon-mediated BAC transgenesis in zebrafish and mice. BMC Genomics 10, 477 (2009).
Shmerling, D. et al. Strong and ubiquitous expression of transgenes targeted into the β-actin locus by Cre/lox cassette replacement. Genes 42, 229–235 (2005).
Tasic, B. et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc. Natl Acad. Sci. USA 108, 7902–7907 (2011).
Ohtsuka, M. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015).
Doetschman, T. et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576–578 (1987).
Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987).
DeChiara, T. M. et al. Producing fully ES cell-derived mice from eight-cell stage embryo injections. Methods Enzym. 476, 285–294 (2010).
Artus, J. & Hadjantonakis, A. K. Generation of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos. Methods Mol. Biol. 693, 37–56 (2011).
Gertsenstein, M. et al. Efficient generation of germ line transmitting chimeras from C57BL/6N ES cells by aggregation with outbred host embryos. PLoS ONE 5, e11260 (2010).
Hu, M. et al. Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos. J. Anim. Sci. Biotechnol. 4, 12 (2013).
Bradley, A., Evans, M., Kaufman, M. H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).
Abuin, A., Hansen, G. M. & Zambrowicz, B. Gene trap mutagenesis. Handb. Exp. Pharmacol. 129–147 (2007); https://doi.org/10.1007/978-3-540-35109-2_6
Cervantes, R. B., Stringer, J. R., Shao, C., Tischfield, J. A. & Stambrook, P. J. Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc. Natl Acad. Sci. USA 99, 3586–3590 (2002).
Liu, X. et al. Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev. Dyn. 209, 85–91 (1997).
Longo, L., Bygrave, A., Grosveld, F. G. & Pandolfi, P. P. The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res. 6, 321–328 (1997).
Birling, M.-C. et al. A resource of targeted mutant mouse lines for 5,061 genes. Nat. Genet. 53, 416–419 (2021).
Ying, Q.-L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).
Ying, Q. L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 (2003).
Skarnes, W. C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).
Kern, H. & Zevnik, B. ES cell line establishment. Methods Mol. Biol. 530, 187–204 (2009).
Voss, A. K., Thomas, T. & Gruss, P. Germ line chimeras from female ES cells. Exp. Cell. Res. 230, 45–49 (1997).
Bronson, S. K., Smithies, O. & Mascarello, J. T. High incidence of XXY and XYY males among the offspring of female chimeras from embryonic stem cells. Proc. Natl Acad. Sci. USA 92, 3120–3123 (1995).
Ying, Q. L. & Smith, A. G. Defined conditions for neural commitment and differentiation. Methods Enzym. 365, 327–341 (2003).
Codner, G. F. et al. Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination. Methods 191, 59–67 (2021).
Hu, T., Chitnis, N., Monos, D. & Dinh, A. Next-generation sequencing technologies: an overview. Hum. Immunol. 82, 801–811 (2021).
Codner, G. F. et al. Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction. BMC Cell Biol. 17, 30 (2016).
Auerbach, A. B. et al. Strain-dependent differences in the efficiency of transgenic mouse production. Transgenic Res. 12, 59–69 (2003).
Alcantar, T. M., Wiler, R. & Rairdan, X. Y. Comparison of BALB/c and B6-albino mouse strain blastocysts as hosts for the injection of C57BL6/N-derived C2 embryonic stem cells. Transgenic Res. 25, 527–531 (2016).
Fielder, T. J. et al. Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts. Transgenic Res. 21, 1149–1158 (2012).
Pacholczyk, G., Suhag, R., Mazurek, M., Dederscheck, S. M. & Koni, P. A. Generation of C57BL/6 knockout mice using C3H × BALB/c blastocysts. BioTechniques 44, 413–416 (2008).
Zevnik, B. et al. C57BL/6N albino/agouti mutant mice as embryo donors for efficient germline transmission of C57BL/6 ES cells. PLoS ONE 9, e90570 (2014).
Schuster-Gossler, K. et al. Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. BioTechniques 31, 1022–1026 (2001).
Lemckert, F. A., Sedgwick, J. D. & Körner, H. Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro. Nucleic Acids Res. 25, 917–918 (1997).
Ma, Y. et al. Efficiency comparison of B6(Cg)-Tyrc-2j /J and C57BL/6NTac embryos as hosts for the generation of knockout mice. Transgenic Res. (2021); https://doi.org/10.1007/s11248-021-00248-9
Koentgen, F. et al. Exclusive transmission of embryonic stem cell-derived genome through the mouse germline. Genesis 54, 326–333 (2016).
Tröder, S. E. & Zevnik, B. History of genome editing: from meganucleases to CRISPR. Lab. Anim. https://doi.org/10.1177/0023677221994613 (2021).
Ménoret, S. et al. Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J. 27, 703–711 (2013).
Geurts, A. M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433–433 (2009).
Carbery, I. D. et al. Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451–459 (2010).
Meyer, M., de Angelis, M. H., Wurst, W. & Kühn, R. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl Acad. Sci. USA 107, 15022–15026 (2010).
Wefers, B. et al. Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA. Nat. Protoc. 8, 2355–2379 (2013).
Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 (2011).
Sung, Y. H. et al. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24 (2013).
Panda, S. K. et al. Highly efficient targeted mutagenesis in mice using TALENs. Genetics 195, 703–713 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).
Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).
Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).
Mehravar, M., Shirazi, A., Nazari, M. & Banan, M. Mosaicism in CRISPR/Cas9-mediated genome editing. Dev. Biol. 445, 156–162 (2019).
Anderson, K. R. et al. CRISPR off-target analysis in genetically engineered rats and mice. Nat. Methods 15, 512–514 (2018).
Akcakaya, P. et al. In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561, 416–419 (2018).
Cui, Y., Xu, J., Cheng, M., Liao, X. & Peng, S. Review of CRISPR/Cas9 sgRNA design tools. Interdiscip. Sci. 10, 455–465 (2018).
Kim, N. et al. Prediction of the sequence-specific cleavage activity of Cas9 variants. Nat. Biotechnol. 38, 1328–1336 (2020).
Lee, K. et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR–Cas9 engineering. Elife 6, e25312 (2017).
Bunton-Stasyshyn, R. K., Codner, G. F. & Teboul, L. Screening and validation of genome-edited animals. Lab. Anim. https://doi.org/10.1177/00236772211016922 (2021).
McBeath, E. et al. Rapid evaluation of CRISPR guides and donors for engineering mice. Genes 11, 628 (2020).
Renaud, J.-B. et al. Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep. 14, 2263–2272 (2016).
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).
Miura, H., Quadros, R. M., Gurumurthy, C. B. & Ohtsuka, M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat. Protoc. 13, 195–215 (2018).
Chu, V. T. et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 16, 4 (2016).
Gu, B., Posfai, E., Gertsenstein, M. & Rossant, J. Efficient generation of large-fragment knock-in mouse models using 2-cell (2C)-homologous recombination (HR)-CRISPR. Curr. Protoc. Mouse Biol. 10, e67 (2020).
Hashimoto, M., Yamashita, Y. & Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. https://doi.org/10.1016/j.ydbio.2016.07.017 (2016).
Qin, W. et al. Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200, 423–430 (2015).
Ohtsuka, M. et al. i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 19, 25 (2018).
Mader, S. L., Libal, N. L., Pritchett-Corning, K., Yang, R. & Murphy, S. J. Refining timed pregnancies in two strains of genetically engineered mice. Lab. Anim. 38, 305–310 (2009).
Zintzsch, A. et al. Guidelines on severity assessment and classification of genetically altered mouse and rat lines. Lab. Anim. 51, 573–582 (2017).
Bundesinstitut für Risikobewertung. Severity assessment of genetically altered mice and rats — V2. Recommendation no. 002/2016 by the National Committee (TierSchG). (2016).
Palmiter, R. D. et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. 1982. Biotechnology 24, 429–433 (1992).
Lipinski, M. M. et al. Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system. EMBO J. 20, 3402–3413 (2001).
Naiche, L. A. & Papaioannou, V. E. Cre activity causes widespread apoptosis and lethal anemia during embryonic development. Genesis 45, 768–775 (2007).
Lexow, J., Poggioli, T., Sarathchandra, P., Santini, M. P. & Rosenthal, N. Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver. Dis. Model Mech. 6, 1470–1476 (2013).
Liptak, N., Bosze, Z. & Hiripi, L. GFP transgenic animals in biomedical research: a review of potential disadvantages. Physiol. Res. 68, 525–530 (2019).
Wefers, B., Wurst, W. & Kühn, R. Design and generation of gene-targeting vectors. Curr. Protoc. Mouse Biol. 1, 199–211 (2011).
FELASA Working Group on Revision of Guidelines for Health Monitoring of Rodents and Rabbits et al. FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab. Anim. 48, 178–192 (2014).
Busch, M. et al. Tiergerechte Haltung von Labormäusen. (GV-SOLAS Society for Laboratory Animal Science, Committee for Humane Laboratory Animal Housing, 2014); https://www.gv-solas.de/wp-content/uploads/2021/08/hal_201408Tiergerechte-Haltung-Maus.pdf
Bahougne, T., Kretz, M., Angelopoulou, E., Jeandidier, N. & Simonneaux, V. Impact of circadian disruption on female mice reproductive function. Endocrinology 161, bqaa028 (2020).
Gaskill, B. N. et al. Energy reallocation to breeding performance through improved nest building in laboratory mice. PLoS ONE 8, e74153 (2013).
Nutrient Requirements of Laboratory Animals: Fourth Revised Edition, 1995. 4758 (National Academies Press, 1995); https://doi.org/10.17226/4758
Hurst, J. L. & West, R. S. Taming anxiety in laboratory mice. Nat. Methods 7, 825–826 (2010).
Gouveia, K. & Hurst, J. L. Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling. Sci Rep. 7, 44999 (2017).
Gouveia, K. & Hurst, J. L. Reducing mouse anxiety during handling: effect of experience with handling tunnels. PLoS ONE 8, e66401 (2013).
Cederwall, M. Positive reinforcement training for laboratory mice. (Swedish Univ. Agricultural Sciences, 2014); https://www.semanticscholar.org/paper/Positive-reinforcement-training-for-laboratory-mice-Cederwall/bbdd52e182cd3bc8431d4cc5fdff24700f924e76
Saunders, T. 2019 ISTT 3Rs awardee - Pawel Pelczar. ISTT Blog https://www.transtechsociety.org/index.php?src=blog&srctype=detail&blogid=17 (2019).
Suthersan, D., Kennedy, S. & Chapman, M. Physical symptoms throughout IVF cycles. Hum. Fertil. 14, 122–128 (2011).
Byers, S. L., Payson, S. J. & Taft, R. A. Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs). Theriogenology 65, 1716–1726 (2006).
Luo, C. et al. Superovulation strategies for 6 commonly used mouse strains. J. Am. Assoc. Lab. Anim. Sci. 50, 471–478 (2011).
Takeo, T. & Nakagata, N. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLoS ONE 10, e0128330 (2015).
Manteca Vilanova, X., De Briyne, N., Beaver, B. & Turner, P. V. Horse welfare during equine chorionic gonadotropin (eCG) production. Animals 9, 1053 (2019).
Hasegawa, A. et al. High-yield superovulation in adult mice by anti-inhibin serum treatment combined with estrous cycle synchronization. Biol. Reprod. 94, 21 (2016).
Crispo, M., Meikle, M. N., Schlapp, G. & Menchaca, A. Ovarian superstimulatory response and embryo development using a new recombinant glycoprotein with eCG-like activity in mice. Theriogenology 164, 31–35 (2021).
Kolbe, T., Sheety, S., Walter, I., Palme, R. & Rülicke, T. Impact of superovulation and mating on the wellbeing of juvenile and adult C57BL/6N mice. Reprod. Fertil. Dev. 28, 969–973 (2016).
Esmail, M. Y., Qi, P., Connor, A. B., Fox, J. G. & García, A. Generating chimeric mice by using embryos from nonsuperovulated BALB/c mice compared with superovulated BALB/c and albino C57BL/6 mice. J. Am. Assoc. Lab. Anim. Sci. 55, 400–405 (2016).
Diamond, M. Intromission pattern and species vaginal code in relation to induction of pseudopregnancy. Science 169, 995–997 (1970).
Miller, A. L., Wright-Williams, S. L., Flecknell, P. A. & Roughan, J. V. A comparison of abdominal and scrotal approach methods of vasectomy and the influence of analgesic treatment in laboratory mice. Lab. Anim. 46, 304–310 (2012).
Haueter, S. et al. Genetic vasectomy-overexpression of Prm1-EGFP fusion protein in elongating spermatids causes dominant male sterility in mice. Genesis 48, 151–160 (2010).
Garrels, W. et al. Direct comparison of vasectomized males and genetically sterile Gapdhs knockout males for the induction of pseudopregnancy in mice. Lab. Anim. 52, 365–372 (2018).
Preece, C. et al. Replacement of surgical vasectomy through the use of wild-type sterile hybrids. Lab. Anim. 50, 49–52 (2021).
Program and Abstracts of the 15th Transgenic Technology Meeting (TT2019). Transgenic Res. 28 (Suppl. 1), 1–33 (2019).
Byers, S. L., Wiles, M. V., Dunn, S. L. & Taft, R. A. Mouse estrous cycle identification tool and images. PLoS ONE 7, e35538 (2012).
Whitten, W. K. Modification of the oestrous cycle of the mouse by external stimuli associated with the male; changes in the oestrous cycle determined by vaginal smears. J. Endocrinol. 17, 307–313 (1958).
Steele, K. H. et al. Nonsurgical embryo transfer device compared with surgery for embryo transfer in mice. J. Am. Assoc. Lab. Anim. Sci. 52, 17–21 (2013).
Green, M. A., Bass, S. & Spear, B. T. A device for the simple and rapid transcervical transfer of mouse embryos eliminates the need for surgery and potential post-operative complications. Biotechniques 47, 919–924 (2009).
Bin Ali, R. et al. Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer. Transgenic Res. 23, 691–695 (2014).
Kolbe, T., Palme, R., Touma, C. & Rülicke, T. Repeated use of surrogate mothers for embryo transfer in the mouse. Biol. Reprod. 86, 1–6 (2012).
Koutroli, E. et al. Effects of using the analgesic tramadol in mice undergoing embryo transfer surgery. Lab. Anim. 43, 167–172 (2014).
Schlapp, G., Goyeneche, L., Fernandez, G., Menchaca, A. & Crispo, M. Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice. J. Assist. Reprod. Genet. 32, 271–275 (2015).
Krueger, K. L. & Fujiwara, Y. The use of buprenorphine as an analgesic after rodent embryo transfer. Lab. Anim. 37, 87–90 (2008).
Goulding, D. R. et al. The effects of perioperative analgesia on litter size in Crl:CD1(ICR) mice undergoing embryo transfer. J. Am. Assoc. Lab. Anim. Sci. 49, 423–426 (2010).
Arras, M. et al. Pain Management for laboratory animals. (GV-SOLAS Society for Laboratory Animal Science Committee for Anaesthesia, 2020); https://www.gv-solas.de/wp-content/uploads/2021/08/2021-04_Pain_Management_for_laboratory_animals.pdf
Norton, W. B. et al. Refinements for embryo implantation surgery in the mouse: comparison of injectable and inhalant anesthesias—tribromoethanol, ketamine and isoflurane—on pregnancy and pup survival. Lab. Anim. 50, 335–343 (2016).
Bagis, H., Odaman Mercan, H. & Dinnyes, A. Exposure to warmer postoperative temperatures reduces hypothermia caused by anaesthesia and significantly increases the implantation rate of transferred embryos in the mouse. Lab. Anim. 38, 50–54 (2004).
Wixson, S. K., White, W. J., Hughes, H. C., Lang, C. M. & Marshall, W. K. The effects of pentobarbital, fentanyl-droperidol, ketamine-xylazine and ketamine-diazepam on core and surface body temperature regulation in adult male rats. Lab. Anim. Sci. 37, 743–749 (1987).
Langford, D. J. et al. Coding of facial expressions of pain in the laboratory mouse. Nat. Methods 7, 447–449 (2010).
Leach, M. C. et al. The assessment of post-vasectomy pain in mice using behaviour and the Mouse Grimace Scale. PLoS ONE 7, e35656 (2012).
Hawkins, P. et al. A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Lab. Anim. 45, 1–13 (2011).
Scarborough, J. et al. Preclinical validation of the micropipette-guided drug administration (MDA) method in the maternal immune activation model of neurodevelopmental disorders. Brain Behav. Immun. 88, 461–470 (2020).
Evangelista-Vaz, R., Bergadano, A., Arras, M. & Jirkof, P. D. Analgesic efficacy of subcutaneous-oral dosage of tramadol after surgery in C57BL/6J mice. J. Am. Assoc. Lab. Anim. Sci. https://doi.org/10.30802/AALAS-JAALAS-17-000118 (2018).
Ingrao, J. C. et al. Aqueous stability and oral pharmacokinetics of meloxicam and carprofen in male C57BL/6 mice. J. Am. Assoc. Lab. Anim. Sci. 52, 553–559 (2013).
Wells, D. J. et al. Assessing the welfare of genetically altered mice. Lab. Anim. 40, 111–114 (2006).
Galichet, C. & Lovell-Badge, R. Applications of genome editing on laboratory animals. Lab. Anim. https://doi.org/10.1177/0023677221993141 (2021).
Okano, H. & Kishi, N. Investigation of brain science and neurological/psychiatric disorders using genetically modified non-human primates. Curr. Opin. Neurobiol. 50, 1–6 (2018).
Eggel, M. & Walker, R. Replacement or reduction of gene-edited animals in biomedical research: a comparative ethics and policy analysis. N. C. Law Rev. 97, 1241 (2019).
Devolder, K. & Eggel, M. No pain, no gain? In defence of genetically disenhancing (most) research animals. Animals 9, 154 (2019).
This Review has been prepared on behalf of the Committee on Genetics and Breeding of the German Society for Laboratory Animal Science (GV-SOLAS). We especially thank our committee colleagues N. Baumgart, J. Davidson, F. Iglauer, S. Nagel-Riedasch, I. Renner-Müller and J. Schenkel for their multiple inputs, discussions and corrections. We would like to thank T. Hennek, I. Hermans-Borgmeyer, T. Kolbe, G. Michels, R. Naumann, J. Parker-Thornburg, T. Rülicke, T. Saunders, S. Sonntag and S. Tröder for their very helpful review and comments. We are grateful to A. Bergadano and P. Jirkof for supporting us with their expertise on analgesic regimens.
The authors declare no competing interests.
Peer review information
Lab Animal thanks Lydia Teboul and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Zevnik, B., Jerchow, B. & Buch, T. 3R measures in facilities for the production of genetically modified rodents. Lab Anim 51, 162–177 (2022). https://doi.org/10.1038/s41684-022-00978-1