Pronuclear Injection‐Based Targeted Transgenesis

Samantha L.P. Schilit1, Masato Ohtsuka2, Rolen M. Quadros3, Channabasavaiah B. Gurumurthy4

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, 3 Mouse Genome Engineering Core Facility, University of Nebraska Medical Center, Omaha, Nebraska, 4 Developmental Neuroscience, Munroe Meyer Institute, University of Nebraska Medical Center, Omaha, Nebraska
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 15.10
DOI:  10.1002/cphg.23
Online Posting Date:  October, 2016
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Abstract

Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy number, or integration into heterochromatic regions that inhibit transgene expression. A recently developed method addresses such pitfalls of traditional transgenesis by targeting the transgene to predetermined sites in the genome that can safely harbor exogenous DNA. This method, called Pronuclear Injection‐based Targeted Transgenesis (PITT), employs an enzymatic transfer of exogenous DNA from a donor vector to a previously created landing‐pad site in the mouse genome. DNA transfer is achieved using molecular tools such as the Cre‐LoxP recombinase and PhiC31‐attB/P integrase systems. Here, we provide protocols for performing PITT and an overview of the current PITT tools available to the research community. © 2016 by John Wiley & Sons, Inc.

Keywords: pronuclear injection; targeted transgenesis; PITT; Cre‐LoxP recombination; PhiC31‐attB/P integration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Designing and Building of PITT Donor DNA Constructs
  • Basic Protocol 2: Synthesis and Purification of DNA and RNA Components for Microinjection
  • Basic Protocol 3: Isolation of Embryos From Seed Mice, Microinjection of PITT Components, and Embryo Transfer
  • Support Protocol 1: Production of Fertilized Eggs Through In Vitro Fertilization (IVF)
  • Basic Protocol 4: Genotyping of Offspring to Identify Transgenic Founders
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Designing and Building of PITT Donor DNA Constructs

  Materials
  • Donor vector backbone (selected from Table 15.10.2)
  • QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)
  • Low‐melt agarose, e.g., SeaPlaque GTG Agarose (Lonza, cat. no. 50111)
  • Modified TE: 10 mM Tris, 0.1 mM EDTA, pH 8.0 (e.g., Affymetrix, cat. no. 75793)
  • Alkaline phosphatase, Calf Intestinal (CIP; NEB, cat. no. M0290)
  • Phenol, TE‐saturated (e.g., Sigma‐Aldrich, cat. no. 77607 or Nacalai Tesque, cat. no. 26829‐54)
  • 3 M sodium acetate (NaOAc) buffer solution, pH 5.2 (e.g., Sigma Aldrich, cat. no. S7899 or Nacalai Tesque, cat. no. 31150‐64)
  • 100% and 70% (v/v) ethanol (Decon Laboratories, cat. no. 07‐678‐005)
  • High fidelity DNA polymerase, e.g., Phusion (NEB, cat. no. M0530) or KOD‐Plus (Toyobo, cat. no. F0934K)
  • Primers with appropriate sequence extensions, to amplify the desired cDNA or expression cassette while adding flanking sequences for cloning
  • Quick Ligation Kit (NEB, cat. no. M2200)
  • Gibson Assembly Master Mix (NEB, cat. no. E2611)
  • Competent cells, e.g., NEB 5‐alpha competent E. coli, high efficiency (NEB, cat. no. C2987)
  • LB broth with agar (Sigma‐Aldrich, cat. no. L3147), containing appropriate antibiotics (e.g., 100 μg/ml ampicillin or 25 μg/ml kanamycin)
  • LB medium (MP Biomedicals, cat. no. 113002022) with appropriate antibiotics
  • Plasmid Mini Kit (Qiagen, cat. no. 12125)
  • Primers for sequencing donor vector
  • Additional reagents and equipment for the extraction and precipitation of DNA ( appendix 3C), polymerase chain reaction (Kramer and Coen, ), agarose gel electrophoresis (unit 2.7; Jarco et al., ), and bacterial transformation. unit 2.7 does not include anything on PCR.

Basic Protocol 2: Synthesis and Purification of DNA and RNA Components for Microinjection

  Materials (see also protocol 1)
  • Donor vector, transformed into E. coli (from protocol 1)
  • Recombinase/integrase in vitro transcription vector, transformed into E. coli
  • Cre platform: pBBI, vector for iCre mRNA synthesis (Addgene, cat. no. 65795; https://www.addgene.org/65795/; Ohtsuka et al., )
  • PhiC31 platform: pBBK, vector for PhiC31o mRNA synthesis (Addgene, cat. no. 62670; https://www.addgene.org/62670/; Ohtsuka et al., )
  • HiSpeed Plasmid Midi Kit (Qiagen, cat. no. 12643)
  • Ultrafree‐MC filter, 0.45‐μm pore size (Millipore, cat. no. UFC30HV00)
  • Quant‐iT PicoGreen dsDNA Assay Kit (Invitrogen, cat. no. P11496)
  • XbaI (NEB, cat. no. R0145), to linearize mRNA synthesis vector
  • Nuclease‐free water (Ambion, cat. no. AM9937), not DEPC‐treated
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol, pH 7.9 (e.g., Nacalai Tesque, cat. no. 25970‐14)
  • Chloroform (e.g., Nacalai tesque, cat. no. 08402‐55)
  • mMESSAGE mMACHINE T7 ULTRA Kit (Ambion, cat. no. AM1345)
  • MEGAclear Transcription Clean‐Up Kit (Ambion, cat. no. AM1908)
  • Microinjection buffer (see recipe)
  • 1.5‐ml RNase‐free microcentrifuge tubes
  • NanoDrop spectrophotometer or fluorescence microplate reader
  • Additional reagents and equipment for the spectroscopic quantification of nucleic acids ( appendix 3D; Gallagher and Desjardins, )

Basic Protocol 3: Isolation of Embryos From Seed Mice, Microinjection of PITT Components, and Embryo Transfer

  Materials
  • Mice: A list of seed mouse strains can be found in Table 15.10.3.
  • Egg donors: wild‐type C57BL/6 female mice procured at 3‐weeks old (Charles River Laboratories, Wilmington, MA).Homozygous seed female mice (bred in‐house), instead of wild type females, can also be used. However, we have found that hemizygous embryos survive better than homozygous embryos after microinjection.
  • Stud males: homozygous seed mice, 3‐ to 6‐months old
  • Pseudo‐pregnant recipients: Crl:CD1(ICR) female mice, 8‐ to 12‐weeks old (purchased at 5‐ to 6‐weeks old; Charles River Laboratories, Wilmington, MA)
  • Vasectomized males: CD1 male mice purchased at 5‐ to 6‐weeks old (Charles River Laboratories, Wilmington, MA) and vasectomized as previously described (Behringer et al., )The vasectomized mice can be used from 6 months to 1 year of age.
  • 2 IU/μl pregnant mare's serum gonadotropin (PMSG; National Hormone and Peptide Program, Harbor–UCLA Medical Center, Torrance, CA)
  • 1 IU/μl human chorionic gonadotropin (hCG; National Hormone and Peptide Program, Harbor–UCLA Medical Center, Torrance, CA)Lyophilized PMSG and hCG are supplied by National Hormone and Peptide Program in vials containing 2000 IU and 1000 IU, respectively. Upon first use, reconstitute in PBS (Millipore, cat. no. BSS‐1006‐B) to a final concentration of 100 IU/100 µl. Prepare 100‐μl aliquots of this 20× stock solution and store at −80°C.
  • 1× EmbryoMax M2 medium with phenol red (Millipore, cat. no. MR‐015‐D), for embryo handling and microinjection
  • 1× EmbryoMax M2 medium with phenol red and hyaluronidase (Millipore, cat. no. MR‐051‐F), for dissociation of the cumulus oophorus complex
  • 1× EmbryoMax KSOM medium with ½ amino acids (Millipore, cat. no. MR‐106‐D), for embryo incubation
  • Microinjection buffer (see recipe)
  • EmbryoMax filtered light mineral oil (Millipore, cat. no. ES‐005‐C)
  • Individually ventilated cages (Allentown, Lab Products, or Tecniplast)
  • 35 × 10‐mm Falcon tissue culture dish (Corning, cat. no. 353001)
  • Falcon IVF/organ culture dish (Corning, cat. no. 353653)
  • Flexipet pipette, 130‐µm (Cook Medical, cat. no. K‐FPIP‐1130‐10BS‐5), for collecting embryos
  • Glass capillaries, 4‐in × 1‐mm (World Precision Instruments, cat. no. TW100F‐4)
  • MicroFil 28‐gauge × 97‐mm long (World Precision Instruments, cat. no. MF28G)
  • 1‐ml subcutaneous syringe and needle (BD, cat. no. 309597)
  • 150 × 15‐mm tissue culture dish (Falcon, cat. no. 351058)
  • Nunc Lab‐Tek chamber slide system (Lab‐Tek, cat. no. 177372)
  • Holding micropipets (Origio, cat. no. MPH‐SM‐20)
  • Heraeus HERAcell 150i Tri–gas incubator, equipped with Coda Inline filters
  • Large slide warmer (Spectrum Scientifics, cat. no. 3875)
  • Dissecting scope: e.g., Leica MZ 9.5 with Plan 0.5× condenser lens (model 10 446 157), base (model 10 445 367), and tilt head
  • Heating glass (Live Cell Instrument, cat. no. HG‐T‐Z002) with temperature controller (Live Cell Instrument, model CU‐301)
  • Mouth pipetting apparatus (assembled as described by Gurumurthy et al., ).
  • Glass micropipette puller (Sutter Instrument Co., model P97), outfitted with a 2.5 × 2.5‐mm box filament (cat. no. FB255B)
  • Microinjection scope (example below) equipped with Narishige IM‐300 microinjector, NT‐88‐V3 manipulator Nikon Eclipse TE 2000‐E with DIC, equipped with Narishige IM 300 microinjector and NT‐88‐V3 micromanipulators, and Live Cell Instrument HG‐T‐Z002 heating glass with CU‐301 temperature controller
  • Leica DM IRB with HC PLAN 10×/22 adjustable eyepiece (cat. no. 11 507 804), S70/0.30 condenser lens, C PLAN 4×/0.10 (cat. no. 11 506 074), N PLAN L 20×/0.40 CORR (cat. no. 11 506 057), N PLAN L 40×/0.55 CORR (cat. no. 11 506 059) objectives
  • Additional reagents and equipment for mouse husbandry, euthanasia, and surgery ( appendix 3L; Donovan et al., ) and transgenesis (Behringer et al., ; Pease and Saunders, )
IMPORTANT: Be sure all animal methods are approved by your institutional animal care and use committee (IACUC).

Support Protocol 1: Production of Fertilized Eggs Through In Vitro Fertilization (IVF)

  Materials (also see protocol 3)
  • Mice:
  • Stud males: homozygous seed mice, 3‐ to 6‐months old (see Table 15.10.3)
  • Egg donors: wild type C57BL/6 female mice procured at 7‐weeks old (CLEA Japan, Inc., Tokyo, Japan).Homozygous seed female mice (bred in‐house), instead of wild type females, can also be used. However, we have found that hemizygous embryos survive better than homozygous embryos after microinjection.
  • PMSG (ASKA Animal Health Co., Ltd., Tokyo, Japan), 1000 IU/ampule, 10 ampules
  • hCG (ASKA Animal Health Co., Ltd., Tokyo, Japan), 3000 IU/ampule, 10 ampules The PMSG and hCG hormones from protocol 3 should be compatible with protocol 4Support Protocol.Reconstitute lyophilized PMSG and hCG in saline (0.9% sodium chloride solution; Otsuka Normal Saline, Otsuka Pharmaceutical Factory, Inc.) to a final concentration of 7.5 IU/0.2 ml and store at −80°C until use.
  • HTF medium (Human Tubal Fluid; ARK Resource, Kumamoto, Japan; http://www.ark‐resource.co.jp/english/products/pr_vitrification)
  • 1× EmbryoMax M2 medium with phenol red (Millipore, cat. no. MR‐015‐D), for embryo handling and microinjection
  • Liquid paraffin (Nacalai Tesque, cat. no. 26137‐85)
  • 35 × 10‐mm Falcon tissue culture dish (Corning, cat. no. 353001)
  • 1‐ml tuberculin syringe with 26G × 0.5‐in needle (TERUMO, cat. no. SS‐01T2613S)
  • Dissecting microscope (e.g., Olympus SZ11) equipped with a hot plate (e.g., KM‐1, Kitazato)
  • Heraeus HERAcell 150i Tri–gas incubator, equipped with Coda Inline filters
IMPORTANT: Be sure all animal methods are approved by your institutional animal care and use committee (IACUC).

Basic Protocol 4: Genotyping of Offspring to Identify Transgenic Founders

  Materials (also see protocol 1)
  • Mice: potential transgenic founders (from protocol 3) and wild‐type controls
  • Cell lysis solution (Qiagen, cat. no. 158908)
  • Proteinase K, 20 mg/ml (5 PRIME, cat. no. 2500150)
  • Protein precipitation solution (Qiagen, cat. no. 158912)
  • 100% and 70% (v/v) ethanol (e.g., Decon Laboratories, cat. no. 07‐678‐005)
  • DNA hydration solution (Qiagen, cat. no. 158914)
  • PCR 2× master mix: e.g., GoTaq Hot Start Green Master Mix (Promega, cat. no. M5122)
  • Nuclease‐free water (such as from Thermo Fisher Scientific, cat. no. BP561‐1)
  • Primer mix: combine equal volumes of forward and reverse primers from 100 pmol/µl stocks, resulting in a final mix containing 50 pmol/µl each primer. Use 1 µl primer mix in 100 µl of PCR master mix.
  • Heat block
  • Vortex mixer
  • Microcentrifuge
  • Thermocycler (BioRad T100 or equivalent)
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Figures

Videos

Literature Cited

  Auerbach, A.B., Norinsky, R., Ho, W., Losos, K., Guo, Q., Chatterjee, S., and Joyner, A.L. 2003. Strain‐dependent differences in the efficiency of transgenic mouse production. Transgenic Res. 12:59‐69. doi: 10.1023/A:1022166921766.
  Behringer, R., Gertsenstein, M., Nagy, K.V., and Nagy, A. 2014. Manipulating the mouse embryo: A laboratory manual. Fourth edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Donovan, J., Brown, P., Reeves, J. and Reeves, P. 2003. Introduction to basic mouse handling techniques. Curr. Protoc. Hum. Genet. 36:A.3L.1–A.3L.18. doi: 10.1002/0471142905.hga03ls36.
  Fielder, T.J., Barrios, L., and Montoliu, L. 2010. A survey to establish performance standards for the production of transgenic mice. Transgenic Res. 19:675‐681. doi: 10.1007/s11248‐009‐9335‐3.
  Gallagher, S. R. and Desjardins, P. R. 2007. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Hum. Genet. 53:A.3D.1–A.3D.21. doi: 10.1002/0471142905.hga03ds53.
  Gordon, J.W. and Ruddle, F.H. 1981. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214:1244‐1246. doi: 10.1126/science.6272397.
  Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., and Ruddle, F.H. 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77:7380‐7384. doi: 10.1073/pnas.77.12.7380.
  Gossler, A., Doetschman, T., Korn, R., Serfling, E., and Kemler, R. 1986. Transgenesis by means of blastocyst‐derived embryonic stem cell lines. Proc. Natl. Acad. Sci. U.S.A. 83:9065‐9069. doi: 10.1073/pnas.83.23.9065.
  Gurumurthy, C.B., Takahashi, G., Wada, K., Miura, H., Sato, M., and Ohtsuka, M. 2016. GONAD: A novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of embryos. Curr. Protoc. Hum. Genet. 88:15.8.1‐15.8.12. doi.wiley.com/10.1002/0471142905.hg1508s88.
  Harms, D.W., Quadros, R.M., Seruggia, D., Ohtsuka, M., Takahashi, G., Montoliu, L., and Gurumurthy, C.B. 2014. Mouse genome editing using the CRISPR/Cas system. Curr. Protoc. Hum. Genet. 83:15.7.1‐15.7.27. doi: 10.1002/0471142905.hg1507s83.
  Hogan, B. 1983. Molecular biology. Enhancers, chromosome position effects, and transgenic mice. Nature 306:313‐314. doi: 10.1038/306313a0.
  Jarcho, J. 2001. Restriction fragment length polymorphism analysis. Curr. Protoc. Hum. Genet. 1:2.7.1‐2.7.15. doi: 10.1002/0471142905.hg0207s01.
  Jones, D. 2011. Genetic engineering of a mouse: Dr. Frank Ruddle and somatic cell genetics. Yale J. Biol. Med. 84:117‐124.
  Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
  Madisen, L., Garner, A.R., Shimaoka, D., Chuong, A.S., Klapoetke, N.C., Li, L., van der Bourg, A., Niino, Y., Egolf, L., Monetti, C., Gu, H., Mills, M., Cheng, A., Tasic, B., Nguyen, T.N., Sunkin, S.M., Benucci, A., Nagy, A., Miyawaki, A., Helmchen, F., Empson, R.M., Knöpfel, T., Boyden, E.S., Reid, R.C., Carandini, M., and Zeng, H. 2015. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85:942‐958. doi: 10.1016/j.neuron.2015.02.022.
  Miura, H., Inoko, H., Tanaka, M., Nakaoka, H., Kimura, M., Gurumurthy, C.B., Sato, M., and Ohtsuka, M. 2015. Assessment of artificial miRNA architectures for higher knockdown efficiencies without the undesired effects in mice. PloS ONE 10:e0135919. doi: 10.1371/journal.pone.0135919.
  Ohtsuka, M. 2014. Development of pronuclear injection‐based targeted transgenesis in mice through Cre‐loxP site‐specific recombination. Methods Mol. Biol. 1194:3‐19. doi: 10.1007/978‐1‐4939‐1215‐5_1.
  Ohtsuka, M., Miura, H., Nakaoka, H., Kimura, M., Sato, M., and Inoko, H. 2012a. Targeted transgenesis through pronuclear injection of improved vectors into in vitro fertilized eggs. Transgenic Res 21:225‐226. doi: 10.1007/s11248‐011‐9505‐y.
  Ohtsuka, M., Miura, H., Sato, M., Kimura, M., Inoko, H., and Gurumurthy, C.B. 2012b. PITT: Pronuclear injection‐based targeted transgenesis, a reliable transgene expression method in mice. Exp. Anim. 61:489‐502. doi: 10.1538/expanim.61.489.
  Ohtsuka, M., Miura, H., Hayashi, H., Nakaoka, H., Kimura, M., Sato, M., Gurumurthy, C.B., and Inoko, H. 2013. Improvement of pronuclear injection‐based targeted transgenesis (PITT) by iCre mRNA‐mediated site‐specific recombination. Transgenic Res. 22:873‐875. doi: 10.1007/s11248‐013‐9703‐x.
  Ohtsuka, M., Ogiwara, S., Miura, H., Mizutani, A., Warita, T., Sato, M., Imai, K., Hozumi, K., Sato, T., Tanaka, M., Kimura, M., and Inoko, H. 2010. Pronuclear injection‐based mouse targeted transgenesis for reproducible and highly efficient transgene expression. Nucleic Acids Res. 38:e198. doi: 10.1093/nar/gkq860.
  Ohtsuka, M., Miura, H., Mochida, K., Hirose, M., Hasegawa, A., Ogura, A., Mizutani, R., Kimura, M., Isotani, A., Ikawa, M., Sato, M., and Gurumurthy, C.B. 2015. One‐step generation of multiple transgenic mouse lines using an improved Pronuclear Injection‐based Targeted Transgenesis (i‐PITT). BMC Genomics 16:274. doi: 10.1186/s12864‐015‐1432‐5.
  Pease, S. and Saunders, T.L. (eds.). 2011. Advanced protocols for animal transgenesis. Springer‐Verlag, Berlin.
  Quadros, R.M., Harms, D.W., Ohtsuka, M., and Gurumurthy, C.B. 2015. Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system. FEBS Open Bio. 5:191‐197. doi: 10.1016/j.fob.2015.03.003.
  Takahashi, H. and Liu, C. 2010. Archiving and distributing mouse lines by sperm cryopreservation, ivf, and embryo transfer. Methods Enzymol. 476:53‐69. doi: 10.1016/S0076‐6879(10)76004‐3.
  Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen‐Tsai, Y., and Luo, L. 2011. Site‐specific integrase‐mediated transgenesis in mice via pronuclear injection. Proc. Natl. Acad. Sci. U.S.A. 108:7902‐7907. doi: 10.1073/pnas.1019507108.
  Tsuchida, J., Matsusaka, T., Ohtsuka, M., Miura, H., Okuno, Y., Asanuma, K., Nakagawa, T., Yanagita, M., and Mori, K. 2016. Establishment of nephrin reporter mice and its use for chemical screening. PLoS ONE 11:e0157497. doi: 10.1371/journal.pone.0157497.
Key Reference
  Ohtsuka, M. 2014. Development of pronuclear injection‐based targeted transgenesis in mice through Cre‐loxP site‐specific recombination. Methods Mol. Biol. 1194:3‐19. doi: 10.1007/978‐1‐4939‐1215‐5_1.
  Some aspects of this protocol were adapted from this reference.
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