Light up the embryos: knock-in reporter generation for mouse developmental biology

Bin Gu About the author

Abstract

Developmental biology seeks to understand the sophisticated regulated process through which a single cell – a fertilized egg – generates a highly organized organism. The most effective way to reveal the nature of these processes is to follow single cells and cell lineages in real-time. Recent advances in imaging equipment, fluorescent tags and computational tools have made long term multi-color imaging of cells and embryos possible. However, there is still one major challenging for achieving live imaging of mammalian embryos- the generation of embryos carrying reporters that recapitulate the endogenous expression pattern of marker genes. Recent developments of genome editing technology played important roles in enabling efficient generation of reporter mouse models. This mini review discusses recent developments of technologies for efficiently generate knock-in reporter mice and the application of these models in live imaging development. With these developments, we are starting to realize the long-sought promises of realtime analysis of mammalian development.

Keywords:
CRISPR-Cas9; genome editing; knock-in reporter; HDR; embryo

Main text

‘Seeing is believing.’ There is nothing more convincing in developmental biology than a movie that reveal the progress of embryonic development in realtime, such as this one showed here (movie 1). This movie captures the process when a mouse preimplantation embryo develops from a 16-cell embryo to a blastocyst with a frame captured every 20 minutes. Two cell lineages were marked by distinctive fluorescent reporters of lineage marker genes, Cdx2-eGFP (in green) for trophectoderm(TE) and Halo-Sox2 (in red) for inner cell mass(ICM). As you can see, the movie captured many critical processes including, the morphogenetic transition from a solid spherical morula to a blastocyst that contains a central cavity- blastocoel, and the sequential emergence of Cdx2 positive TE and Sox2 positive ICM cells. You can even tell that the Sox2 positive cells emerge roughly 9 hours after the Cdx2 positive cells from the time stamps. With this level of spatio-temporal resolution, developmental biology is entering an exciting time. A few technological advancements are required for developmental biology to enter the live imaging time: First, fluorescent tag proteins need to be developed for visualizing a biological entity such as protein, RNA or DNA in living embryos. There are indeed major advancements recently on this line. A large variety of fluorescent proteins, self-labelling tags and bridging molecules has allowed the visualization of almost any molecular entity of the Central Dogma in living embryos by fluorescent imaging spanning a broad optical spectrum – from blue to infra-red (Tsien, 1998Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67(1):509-44. http://dx.doi.org/10.1146/annurev.biochem.67.1.509. PMid:9759496.
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). Second, new microscopes such as light-sheet microscopy and multiphoton microscopy, which significantly decrease photo-toxicity during imaging, have created unprecedented possibilities for long-term live imaging of embryonic development (Benninger et al., 2008Benninger RK, Hao M, Piston DW. Multi-photon excitation imaging of dynamic processes in living cells and tissues. Rev Physiol Biochem Pharmacol. 2008;160:71-92. http://dx.doi.org/10.1007/112_2008_801. PMid:18418560.
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). Third, advanced computational tools are needed and have been developed to extract biological relevant information from large imaging datasets (Amat et al., 2015Amat F, Hockendorf B, Wan Y, Lemon WC, McDole K, Keller PJ. Efficient processing and analysis of large-scale light-sheet microscopy data. Nat Protoc. 2015;10(11):1679-96. http://dx.doi.org/10.1038/nprot.2015.111. PMid:26426501.
http://dx.doi.org/10.1038/nprot.2015.111...
). However, with of all these advancements, the first challenge we need to overcome for realizing all the promises of studying development in realtime, is still to obtain the embryo that carry the specific fluorescent reporters. It is actually quite challenging to obtain these reporter embryos in an efficient and scalable manner, especially for mammalian species such as mouse. This minireview will focus on the recent developments in the gene editing technology, including some of our own experiences, in efficiently generating knock-in reporter mouse models for live imaging research. This is not meant to be an exhaustive review of literature in knock-in technology, instead I will use a few approaches that have been relatively broadly implemented to illustrate the principles behind the new knock-in technologies to generate reporters. I regret not being able to discuss numerous interesting technology developments in this field.

Scientists have been generating reporter mice for a long time, mostly using two classical technologies. The first one is random transgenesis. In this case, protein dynamics were investigated through the use of fluorescent proteins driven by partial regulatory sequences such as promoter or enhancer regions of a gene-of-interest (Ikawa et al., 1998Ikawa M, Yamada S, Nakanishi T, Okabe M. ‘Green mice’ and their potential usage in biological research. FEBS Lett. 1998;430(1-2):83-7. http://dx.doi.org/10.1016/S0014-5793(98)00593-6. PMid:9678599.
http://dx.doi.org/10.1016/S0014-5793(98)...
). These reporter constructs are integrated randomly into the mouse genome through pronuclear microinjection- a relatively fast process more amenable for large scale production (Ittner and Gotz, 2007Ittner LM, Gotz J. Pronuclear injection for the production of transgenic mice. Nat Protoc. 2007;2(5):1206-15. http://dx.doi.org/10.1038/nprot.2007.145. PMid:17546016.
http://dx.doi.org/10.1038/nprot.2007.145...
). This approach has been instrumental in revealing gene expression patterns in live embryos. However, random transgenesis has intrinsic drawbacks. For example, random transgenic reporters frequently fail to recapitulate gene expression patterns due to the lack of full regulatory sequences. Also, the effects of local chromatin environment of the particular insertion site contribute to the expression variability between lines carrying the same transgene (Palmiter et al., 1983Palmiter RD, Norstedt G, Gelinas RE, Hammer RE, Brinster RL. Metallothionein-human GH fusion genes stimulate growth of mice. Science. 1983;222(4625):809-14. http://dx.doi.org/10.1126/science.6356363. PMid:6356363.
http://dx.doi.org/10.1126/science.635636...
; Laboulaye et al., 2018Laboulaye MA, Duan X, Qiao M, Whitney IE, Sanes JR. Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes. Front Mol Neurosci. 2018;11:385. http://dx.doi.org/10.3389/fnmol.2018.00385. PMid:30405348.
http://dx.doi.org/10.3389/fnmol.2018.003...
; Weis et al., 1992Weis J, Fine SM, Sanes JR. Integration site-dependent transgene expression used to mark subpopulations of cells in vivo: an example from the neuromuscular junction. Brain Pathol. 1992;2(1):31-7. PMid:1341945.). Moreover, recent efforts of mapping the insertion locus of a large number of established transgenic mouse lines has identified numerous accompanying complex genomic changes in addition to the transgene insertion. Such changes include large insertions or deletions at the insertion site, which could lead to confounding phenotypes unrelated to the particular transgene (Goodwin et al., 2019Goodwin LO, Splinter E, Davis TL, Urban R, He H, Braun RE, Chesler EJ, Kumar V, van Min M, Ndukum J, Philip VM, Reinholdt LG, Svenson K, White JK, Sasner M, Lutz C, Murray SA. Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res. 2019;29(3):494-505. http://dx.doi.org/10.1101/gr.233866.117. PMid:30659012.
http://dx.doi.org/10.1101/gr.233866.117...
). Knock-in, defined as the precise insertion of an exogenous sequence into an endogenous genomic locus, could largely resolve these drawbacks and achieve faithful tagging of endogenous genes. Traditionally, knock-in mouse embryonic stem(ES) cells are first generated through homologous recombination mediated gene targeting, which typically has a low success frequency. Then these knock-in ES cells are aggregated with or injected into wildtype mouse preimplantation embryos to establish chimeric founders. Only when the knock-in ES cells contributed to the germline of the chimeric founders, knock-in mouse lines can be established. Therefore, it typically requires the screening of hundreds of ES cell clones, which is followed by time consuming chimera generation and breeding (Thomas and Capecchi, 1987Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51(3):503-12. http://dx.doi.org/10.1016/0092-8674(87)90646-5. PMid:2822260.
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http://dx.doi.org/10.1038/nrg1619...
). It is apparent that neither of these technologies would be sufficient for the precision and scalability of reporter generation if we are going to realize the promising of live imaging in developmental biology.

New genome editing technologies, most notably the CRISPR/Cas9 system, have completely changed the landscape in gene editing (Hsu et al., 2014Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-78. http://dx.doi.org/10.1016/j.cell.2014.05.010. PMid:24906146.
http://dx.doi.org/10.1016/j.cell.2014.05...
). Particularly, it allowed targeted gene editing directly through micro-injecting into early embryos by targeted introduction of double strand breaks at desired genomic locus, which bypasses the typically tedious and time-consuming cell engineering steps (Yang et al., 2013Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154(6):1370-9. http://dx.doi.org/10.1016/j.cell.2013.08.022. PMid:23992847.
http://dx.doi.org/10.1016/j.cell.2013.08...
). CRISPR/Cas9 zygote injection has been proved to be highly efficient in generating knock-out alleles through non-homologous end join(NHEJ) pathway, as well as generating point mutation and small tag insertions through homology directed repair(HDR) (Yang et al., 2013Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154(6):1370-9. http://dx.doi.org/10.1016/j.cell.2013.08.022. PMid:23992847.
http://dx.doi.org/10.1016/j.cell.2013.08...
). These approaches have brought new hope for rapidly generating reporter embryos. However, although with initial promising reports, the efficiency of HDR mediated large fragment knock-in, which is required for inserting most fluorescent tags, through mouse zygote injection tends to be variable and not robust (Yang et al., 2013Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154(6):1370-9. http://dx.doi.org/10.1016/j.cell.2013.08.022. PMid:23992847.
http://dx.doi.org/10.1016/j.cell.2013.08...
; Cohen, 2016Cohen J. ‘Any idiot can do it.’ Genome editor CRISPR could put mutant mice in everyone’s reach. Science. 2016:89407621. http://dx.doi.org/10.1126/science.aal0334.). A few recently developed technologies have improved the efficiency of reporter mice generation.

The first was a technology called Efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) (Quadros et al., 2017Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 2017;18(1):92. http://dx.doi.org/10.1186/s13059-017-1220-4. PMid:28511701.
http://dx.doi.org/10.1186/s13059-017-122...
). Instead of double-stranded plasmid DNA that were traditional used for gene targeting, Easi-CRISPR uses long single-stranded DNA as donor templates (Quadros et al., 2017Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 2017;18(1):92. http://dx.doi.org/10.1186/s13059-017-1220-4. PMid:28511701.
http://dx.doi.org/10.1186/s13059-017-122...
; Miura et al., 2018Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. 2018;13(1):195-215. http://dx.doi.org/10.1038/nprot.2017.153. PMid:29266098.
http://dx.doi.org/10.1038/nprot.2017.153...
). By microinjection of ribonucleoprotein complexes of Cas9 protein bound with a complexed crRNA and tracrRNA guide alongside a long ssDNA template encoding the desired knock-in sequence flanked by 60 to 105 base homology arms, Easi-CRISPR was used to accomplish knock-in of fragments varying from 800 base pairs to 1.4 kilobase pairs with efficiencies ranging from 25-67% (Quadros et al., 2017Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 2017;18(1):92. http://dx.doi.org/10.1186/s13059-017-1220-4. PMid:28511701.
http://dx.doi.org/10.1186/s13059-017-122...
). However, at present, long single-stranded DNA template synthesis remains a primary limitation of Easi-CRISPR, with insertion of fragments greater than 1-2 kilobases remaining a challenge. Furthermore, it has been observed that the secondary structure that may be formed within a long single-stranded DNA molecule could lead to the skipping of donor sequences during repair (Codner et al., 2018Codner GF, Mianne J, Caulder A, Loeffler J, Fell R, King R, Allan AJ, Mackenzie M, Pike FJ, McCabe CV, Christou S, Joynson S, Hutchison M, Stewart ME, Kumar S, Simon MM, Agius L, Anstee QM, Volynski KE, Kullmann DM, Wells S, Teboul L. Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol. 2018;16(1):70. http://dx.doi.org/10.1186/s12915-018-0530-7. PMid:29925374.
http://dx.doi.org/10.1186/s12915-018-053...
). This poses another challenge for precise knock-in using Easi-CRISPR.

A crucial technological advancement – two-cell homologous recombination (2C-HR)-CRISPR – was provided by our group based on insights from the understanding of mouse early embryonic development (Gu et al., 2018Gu B, Posfai E, Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol. 2018;36(7):632-7. http://dx.doi.org/10.1038/nbt.4166. PMid:29889212.
http://dx.doi.org/10.1038/nbt.4166...
). It is well-established that HDR is predominantly active in the late S-G2 phases of the cell cycle (Hustedt and Durocher, 2016Hustedt N, Durocher D. The control of DNA repair by the cell cycle. Nat Cell Biol. 2016;19(1):1-9. http://dx.doi.org/10.1038/ncb3452. PMid:28008184.
http://dx.doi.org/10.1038/ncb3452...
). Indeed, there had been numerous reports that timing genome editing to coincide with this period in vitro could lead to significantly greater frequency of editing events mediated by HDR (Yang et al., 2016Yang D, Scavuzzo MA, Chmielowiec J, Sharp R, Bajic A, Borowiak M. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep. 2016;6(1):21264. http://dx.doi.org/10.1038/srep21264. PMid:26887909.
http://dx.doi.org/10.1038/srep21264...
; Lin et al., 2014Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 2014;3:e04766. http://dx.doi.org/10.7554/eLife.04766. PMid:25497837.
http://dx.doi.org/10.7554/eLife.04766...
). We reasoned that the two-cell stage mouse embryo, which have an exceptionally long G2 phase (10-12 hours), might permit higher knock-in efficiency. Furthermore, the accessible chromatin state during zygotic genome activation, which occurs during the elongated G2 phase of two-cell embryos, could also contribute to efficient knock-in (Svoboda, 2018Svoboda P. Mammalian zygotic genome activation. Semin Cell Dev Biol. 2018;84:118-26. http://dx.doi.org/10.1016/j.semcdb.2017.12.006. PMid:29233752.
http://dx.doi.org/10.1016/j.semcdb.2017....
). Indeed, in a side-by-side experiment, delivery of the editing components to a two-cell stage embryo resulted in a significant enhancement of knock-in efficiency compared to zygotes, increasing the efficiency from 1-5% to 30-35% at the two loci tested. Among many gene locus, 2C-HR-CRISPR allowed for the knock-in of reporter alleles in up to 60% of founder mice – simply by changing the timing of delivery. Importantly, 2C-HR-CRISPR was shown to be highly efficient in spite of knock-in size and was used to introduce fragments as large as 7.5 kilobases. To further improve 2C-HR-CRISPR, we modified the system once further. There are speculations that HDR is hindered by the search for homologous sequences following the introduction of a DSB (Ma et al., 2017Ma M, Zhuang F, Hu X, Wang B, Wen XZ, Ji JF, Xi JJ. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res. 2017;27(4):578-81. http://dx.doi.org/10.1038/cr.2017.29. PMid:28266543.
http://dx.doi.org/10.1038/cr.2017.29...
). Therefore, recruiting the donor template to the DSB-site might increase the efficiency of HDR. One efficient method to recruit DNA template to a Cas9 induced DSB is through the biotin-avidin interaction. Therefore, we constructed a Cas9-monomeric streptavidin(mSA) fusion gene. By co-injecting Cas9-mSA mRNA, sgRNA and a biotinylated PCR product as repair template, the repair template would bind to the Cas9-mSA protein and be recruited to the proximity of the double strand break and improve the knock-in efficiency. With this modification, the 2C-HR-CRISPR system produced knock-in efficiencies of up to 95% in some genes at the founder mouse stage (e.g., Gata6-Halo reporter gene) and high germline transmission – with some targets reaching 100%. Using 2C-HR-CRISPR, we have generated 35 knock-in reporters for critical developmental regulators, and moving forward to understand the developmental dynamics of mouse embryonic development. Some of these mouse models have been used to dissect the cell migration process in mouse gastrulation and the mechanical controls of mouse early limb development (McDole et al., 2018McDole K, Guignard L, Amat F, Berger A, Malandain G, Royer LA, Turaga SC, Branson K, Keller PJ. In Toto Imaging and reconstruction of post-implantation mouse development at the single-cell level. Cell. 2018;175(3):859-76.e33. https://doi.org/10.1016/j.cell.2018.09.031.
https://doi.org/10.1016/j.cell.2018.09.0...
; Zhu et al., 2020Zhu M, Tao H, Samani M, Luo M, Wang X, Hopyan S, Sun Y. Spatial mapping of tissue properties in vivo reveals a 3D stiffness gradient in the mouse limb bud. Proc Natl Acad Sci USA. 2020;117(9):4781-91. http://dx.doi.org/10.1073/pnas.1912656117. PMid:32071242.
http://dx.doi.org/10.1073/pnas.191265611...
).

While the techniques described above can reliably mediate large-fragment knock-in, in each case, their successful implementation all require micro-injection gene editing reagents into mouse early embryos. The technical challenges of micro-injection pose significant hurdle for their broad application. Recently, multiple groups have developed injection-free knock-in methods by delivering gene editing reagents using adeno-associated viral vectors into mouse zygotes (Chen et al., 2019Chen S, Sun S, Moonen D, Lee C, Lee AY, Schaffer DV, He L. CRISPR-READI: efficient generation of knockin mice by CRISPR RNP electroporation and AAV Donor infection. Cell Rep. 2019;27(13):3780-9.e4. https://doi.org/10.1016/j.celrep.2019.05.103.
https://doi.org/10.1016/j.celrep.2019.05...
; Yoon et al., 2018Yoon Y, Wang D, Tai PWL, Riley J, Gao G, Rivera-Perez JA. Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Nat Commun. 2018;9(1):412. http://dx.doi.org/10.1038/s41467-017-02706-7. PMid:29379011.
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, Mizuno et al., 2018Mizuno N, Mizutani E, Sato H, Kasai M, Ogawa A, Suchy F, Yamaguchi T, Nakauchi H. Intra-embryo gene cassette knockin by CRISPR/Cas9-mediated genome editing with adeno-associated viral vector. iScience. 2018;9:286-97. https://doi.org/10.1016/j.isci.2018.10.030.
https://doi.org/10.1016/j.isci.2018.10.0...
). A common theme of these techniques is the delivery of donor templates using AAV vectors, while the Cas9 and sgRNA are delivered either as AAV vectors or as Cas9-sgRNA complex (RNPs). These technologies have been shown to generate knock-in reporters with high efficiency (50%), therefore, greatly simplified the process of generating knock-in reporter mice. However, size of the knock-in cassette is limited by the packaging capability of AAV vector(4.9kb).

Other HDR independent technologies such as the micro-homology mediated end joining (MMEJ) mediated CRISPR/Cas9-based precise integration into the targeted chromosome (CRIS-PITCh) and homology mediated end joining (HMEJ) based methods has also been developed to generate knock-in reporters in a wide range of organisms (Nakade et al., 2014Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki KT. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun. 2014;5(1):5560. http://dx.doi.org/10.1038/ncomms6560. PMid:25410609.
http://dx.doi.org/10.1038/ncomms6560...
; Yao et al., 2017Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang P, Sun Q, Shi L, Yang H. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27(6):801-14. http://dx.doi.org/10.1038/cr.2017.76. PMid:28524166.
http://dx.doi.org/10.1038/cr.2017.76...
). These technologies as well hold great potential in generating knock-in reporter mice, although they are right now less used in mouse genetics, probably because the HDR based methods, that typically enable more sequence precision, are sufficiently efficient for mouse generation.

With these exciting technology developments, we are already seeing a number of reported studies dissecting mouse embryonic development using live imaging. With more and more knock-in reporters generated and characterized, we will enter a bright future of mammalian developmental biology.

  • Erratum

    In the article entitle “Light up the embryos: knock-in reporter generation for mouse developmental biology”, DOI number: https://doi.org/10.1590/1984-3143-AR2020-0055, published in journal Animal Reproduction, 2020, volume 17, number 3, page 1:
    Where it reads:
    “Thematic Section: 36th Annual Meeting of the Association of Embryo Technology in Europe (AETE)”
    It should read:
    “Thematic Section: 34th Annual Meeting of the Brazilian Embryo Technology Society (SBTE)”
  • Financial support: None.
  • How to cite: Gu B. Light up the embryos: knock-in reporter generation for mouse developmental biology. Anim Reprod. 2020;17(3):e20200055. https://doi.org/10.1590/1984-3143-AR2020-0055

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Publication Dates

  • Publication in this collection
    10 Aug 2020
  • Date of issue
    2020

History

  • Received
    15 May 2020
  • Accepted
    26 June 2020
Colégio Brasileiro de Reprodução Animal Coronel José dias Bicalho, 1224, CEP: , 31275-050, Belo Horizonte, MG - Brasil, Tel.: 55-31-3491 7122 - Belo Horizonte - MG - Brazil
E-mail: animreprod@cbra.org.br