in vivo MO in mouse?
Sarah S
Thursday, 22 January 2009 20:32 UTC
I have used some MOs in cell culture lines with some sucess. However, I would really like to find someone with experince in using MOs in mouse embryos. The KO mouse for my gene of interest died in utero . I would like to selectively block the 2 splice forms that are expressed only in mature myocytes and find their function. We have not had much luck on the zebrafish front since we have not been able to make good antibodies to the different zebrafish isoforms. I have a collaborator willing to be involved in thsi project but he does not have experience with MOs. Any one out there interested in helping?
thanks
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Replies
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Please, respond to Sarah!
I worry my response might stop others, but I am no help as a collaborator for mouse oocyte/embryo studies. However, I can offer some information and feel free to call me at Gene Tools.
Here are a few citations to papers reporting use of Morpholinos in mouse oocytes or embryos. A new technology you might consider is the Vivo-Morpholino, but there are no publications describing use of Vivo-Morpholinos for mouse embryo – yet.
Coonrod SA, Bolling LC, Wright PW, Visconti PE, Herr JC. A morpholino phenocopy of the mouse MOS mutation. Genesis. 2001 Jul;30(3):198-200.
Lefebvre C, Terret ME, Djiane A, Rassinier P, Maro B, Verlhac MH. Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate. J Cell Biol. 2002 May 13;157(4):603-13.
Mellitzer G, Hallonet M, Chen L, Ang S. Spatial and temporal ‘knock down’ of gene expression by electroporation of double-stranded RNA and morpholinos into early postimplantation mouse embryos. Mech Dev. 2002 Oct;118(1-2):57.
Siddall LS, Barcroft LC, Watson AJ. Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides. Mol Reprod Dev. 2002 Dec;63(4):413-421.
O’Hara MF, Nibbio BJ, Craig RC, Nemeth KR, Charlap JH, Knudsen TB. Mitochondrial benzodiazepine receptors regulate oxygen homeostasis in the early mouse embryo. Reprod Toxicol. 2003 Jul-Aug;17(4):365-75.
Kanzler B, Haas-Assenbaum A, Haas I, Morawiec L, Huber E, Boehm T. Morpholino oligonucleotide-triggered knockdown reveals a role for maternal E-cadherin during early mouse development. Mech Dev. 2003 Dec;120(12):1423-1432.
Cheng TC, Huang CC, Chen CI, Liu CH, Hsieh YS, Huang CY, Lee MS, Liu JY. Leukemia Inhibitory Factor Antisense Oligonucleotide Inhibits the Development of Murine Embryos at Preimplantation Stages. Biol Reprod. 2004 May;70(5):1270-6. Epub 2003 Dec 26.
Mazerbourg S, Hsueh AJ. Growth differentiation factor-9 signaling in the ovary. Mol Cell Endocrinol. 2003 Apr 28;202(1-2):31-6.
Homer HA, McDougall A, Levasseur M, Herbert M. Restaging the Spindle Assembly Checkpoint in Female Mammalian Meiosis I. Cell Cycle. 2005 May;4(5):650-3. Epub 2005 May 14.
Homer HA, McDougall A, Levasseur M, Yallop K, Murdoch AP, Herbert M. Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes. Genes Dev. 2005 Jan 15;19(2):202-7.
Hyenne V, Louvet-Vallee S, El-Amraoui A, Petit C, Maro B, Simmler MC. Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis. Dev Biol. 2005 Sep 28; [Epub ahead of print]
Homer HA, McDougall A, Levasseur M, Murdoch AP, Herbert M. Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes. Reproduction. 2005 Dec;130(6):829-43.
Beraldi R, Pittoggi C, Sciamanna I, Mattei E, Spadafora C. Expression of LINE-1 retroposons is essential for murine preimplantation development. Mol Reprod Dev. 2006 Mar;73(3):279-87.
Tsuji I, Mitani T, Mitsuhashi A, Watanabe Y, Hosoi Y, Hoshiai H. Inhibition of oct4 expression in mouse preimplantation embryos using morpholino antisense oligonucleotides. Tohoku J Exp Med. 2006 Apr;208(4):333-42.
Madgwick S, Hansen DV, Levasseur M, Jackson PK, Jones KT. Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis. J Cell Biol. 2006 Sep 11;174(6):791-801.
Reis A, Chang HY, Levasseur M, Jones KT. APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division. Nat Cell Biol. 2006 May;8(5):539-40.
Marangos P, Verschuren EW, Chen R, Jackson PK, Carroll J. Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APCCdh1. J Cell Biol. 2007 Jan 1;176(1):65-75. Epub 2006 Dec 26.
Furuya M, Tanaka M, Teranishi T, Matsumoto K, Hosoi Y, Saeki K, Ishimoto H, Minegishi K, Iritani A, Yoshimura Y. H1foo is Indispensable for Meiotic Maturation of the Mouse Oocyte. J Reprod Dev. 2007 Aug;53(4):895-902. Epub 2007 May 23.
Reis A, Madgwick S, Chang HY, Nabti I, Levasseur M, Jones KT. Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes. Nat Cell Biol. 2007 Oct;9(10):1192-8. Epub 2007 Sep 23.
Nabti I, Reis A, Levasseur M, Stemmann O, Jones KT. Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs. Dev Biol. 2008 Jul 4. [Epub ahead of print]
Brunet S, Dumont J, Lee KW, Kinoshita K, Hikal P, Gruss OJ, Maro B, Verlhac MH. Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes. PLoS ONE. 2008 Oct 3;3(10):e3338.
Foygel K, Choi B, Jun S, Leong DE, Lee A, Wong CC, Zuo E, Eckart M, Reijo Pera RA, Wong WH, Yao MW. A novel and critical role for Oct4 as a regulator of the maternal-embryonic transition. PLoS ONE. 2008;3(12):e4109. Epub 2008 Dec 31.
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Hi Sarah,
my name is Annett and I am working as a post doc for Mylene Yao at the Stanford University. We established expertise in MO microinjection into the early mouse embryo. We just submitted our protocol to Nature protocol and hopefully it will be online available in the next couple of days. Please look for the title:
“Morpholino-mediated gene knockdown in the early mouse embryo”Denise E. Leong1, Annett Hahn-Windgassen1, Kira Foygel1, Sunny Jun1, Barry Behr1, Mylene W. M. Yao1,*.
1Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.Please let me know if you have specific question, I am more than happy to answer and help you!
Best Annett
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Anne,
Thank you very much for your reply. I am at a small public college, we do not have access to any of the nature journals (can you believe it!?) would yu be able to send me a PDF?thanks!
sspinette@ric.edu
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Congratulations Annett, I am looking forward to seeing your paper!
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Here is the mouse Morpholino protocol from the Nature Protocols site:
http://www.natureprotocols.com/2009/06/05/morpholinomediated_gene_knockd.php“Here we describe a morpholino-based gene knockdown microinjection protocol to interrogate gene function at the maternal-embryonic transition.”
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Hi,
My name is Chanchao, i am working as a postdoc in singapore. I just start working on Mo injection into mouse embryo. I am facing the microinjection technical problems so far, as the cell membrane of Zygote stage embryo is very elastic, it is very difficult (to me) to get into the cytoplasm. Does any one have any trick for microinjection. -
Hi Chanchao,
I hope someone with experience in mouse embryo injection can contribute their experience.
The folks on the Xenopus tropicalis newsgroup are discussing salt concentration and firmness of eggs. I wonder whether you can change the firmness of the mouse zygotes by adjusting the osmolarity of the solution you hold them in while injecting. I’ve not tried this, but if the zygotes survive when temporarily held in a range of osmolarities then this might be a useful approach for adjusting firmness for injection.
Best wishes,
- Jon -
Hi Jon,
thank you so much, i will try.
Chanchao
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