Genetically Engineered Mice by Pronuclear DNA Microinjection

互联网2013-12-31

2231

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The generation of transgenic mice by DNA microinjection is a powerful tool to investigate the molecular regulation of gene expression, development, and disease. The power of this technology is that foreign DNA can be introduced into every cell of a developing organism, and the phenotypic impact of this genetic modification can be investigated in a system under the constraints of normal development and physiology. The generation of transgenic mice requires the preparation of the transgene DNA construction, collection of one?cell fertilized mouse embryos, injection of the transgene into mouse embryos, and transfer of the surviving embryos into a pseudopregnant female. Mice born from such manipulations are then screened for the presence of the transgene. The execution of these procedures requires a highly efficient system; otherwise, the cost of the generation of these mice can be cost prohibitive. However, the production of these animals can provide an invaluable research resource. Curr. Protoc. Mouse Biol. 2:245?262 © 2012 by John Wiley & Sons, Inc.

Keywords: transgenic; mouse; DNA microinjection

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Introduction
Basic Protocol 1: Preparation of Transgene DNA
Basic Protocol 2: Embryo Collection
Basic Protocol 3: Microinjection
Basic Protocol 4: Embryo Transfer
Support Protocol 1: Embryo Culture
Basic Protocol 5: Identification of Transgenic Mice
Reagents and Solutions
Commentary
Literature Cited
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Transgene DNA

Materials

Isolated plasmid DNA or bacterial colony with BAC containing transgene of interest
Restriction enzyme(s) and appropriate reaction buffer(s)
TAE (see recipe )
Agarose (Thermo Fisher Scientific, #BP1356‐500)
10 mg/ml ethidium bromide
GeneClean II kit (MP Biomedicals, www.mpbio.com)
Microinjection TE buffer (see recipe )
LB medium
Antibiotics (for growth of BAC culture)
P1 (50 mM Tris⋅Cl, 10 mM EDTA, pH 8.0). Store at 4°C
P2 (200 mM NaOH, 1% SDS). Store at room temperature
P3 (2.8 M potassium acetate, pH 5.5). Store at 4°C
Ready‐Lyse (Epicentre Biotechnologies)
NucleoBond BAC 100 kit (Clontech)
TE buffer, pH 7.5
7.5 M potassium acetate, autoclaved
Proteinase K
100% ethanol
RNase A
10% SDS
Phenol, buffered, pH 8
Chloroform
7.5 M ammonium acetate
Isopropanol
70% ethanol
Terminase enzyme
3.0 M sodium acetate
BAC microinjection buffer (see recipe )

Gel tray, gel box, and power supply for electrophoresis
Centrifuge bottles
JA‐10 rotor
High‐speed 30‐ml centrifuge tubes
Water bath, 30°C

Basic Protocol 2: Embryo Collection

Materials

3‐ to 4‐week‐old female mice
Pregnant mare serum gonadotropin (PMSG) (VWR International, #80056‐608)
hCG (purchase pregnyl hCG or substitute from local pharmacy)
Breeder male mice
M2 medium (Millipore, #MR‐015‐D)
Hyaluronidase (Sigma‐Aldrich, #H3506‐1G)

Microdissection tools
Embryo transfer pipets (see recipe )

Basic Protocol 3: Microinjection

Materials

DNA from plasmid or BAC ( protocol 1 ) or lentiviral DNA
Paraffin oil
M2 medium (DNA injection) or M16 medium (Millipore #MR‐010P‐5F) (lentivirus injection)
Cytochalasin B stock solution (see recipe )
M2/CB (2.5 µl cytochalasin B stock solution in 497.5 µl M2 medium)
Mouse embryos ( protocol 2 )
Dow Corning 200 Fluid, 50 CST (Ellsworth Adhesives, http://www.ellsworth.com)

Glass disposable micropipets, borosilicate with filament (Sutter Instrument Company #BF100‐78‐10)
Sutter micropipet puller, P1000 or P‐97
Teflon tubing
Injection and holding needle holders
Micrometer syringes for injection and holding needle holders (Stoelting)
Micromanipulators (Narishige or Eppendorf) – Coarse and hydraulic
Inverted microscope (e.g., Nikon, Zeiss)
Disposable micropipets (VWR)
Microforge (Sensaur)
50 × 9‐mm petri dishes with tight lids (Fisher #08‐757‐105)

Basic Protocol 4: Embryo Transfer

Materials

ICR females >24 g
B6D2F1 vasectomized breeder males
1.25% Avertin working solution
Betadine
70% ethanol

Syringe for administration of anesthetic
Hair clippers
Microdissection tools
Embryo transfer pipets (see recipe )
Mouth pipet and tubing for embryo transfers (Renova Life, Inc., #mp‐set and mp 25)
Dissecting microscope
Wound clips and applicator
Heating pad

Support Protocol 1: Embryo Culture

Materials

Appropriate culture medium, such as KSOM, preincubated in CO₂ incubator
Silicon oil (Dow Corning 200 Fluid)
60 × 15 mm culture dishes

Basic Protocol 5: Identification of Transgenic Mice

Materials

1.25% Avertin working solution
TNES (see recipe )
20 mg/ml proteinase K
5 M NaCl
70% and 95% ethanol
TE, pH 7.5

Eartag or ear punch
15‐ or 50‐ml test tubes

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 1. Overall scheme for the generation of genetically engineered mice.

View Image

Figure 2. Embryo collection. (A ) Ampulla of the mouse oviduct being gently torn to release embryos. (B ) Embryos being released from ampulla. (C ) Fertile one‐cell embryos. (D ) Unfertilized ova and fragmented cells.

View Image

Figure 3. Inverted microscope with Hoffman objective lenses and micromanipulators. Holding and injection needles are in place and lowered into injection dish containing embryos.

View Image

Figure 4. Enlarging opening of injection needle by brushing needle against the polished holding needle.

View Image

Figure 5. DNA microinjection into the male pronucleus of the one‐cell mouse embryo.

View Image

Figure 6. DNA microinjection into the male pronucleus of the one‐cell mouse embryo. (A ) Injection needle moving into the male pronucleus of the one‐cell embryo. (B ) Expansion of the male pronucleus upon delivery of DNA under hydrostatic pressure. The black arrows show that the pronucleus diameter increases in size by approximately 50%.

View Image

Videos

Literature Cited

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.
	Brinster, R.L. 1968. In vitro culture of mammalian embryos. J. Anim. Sci. 27:1‐14.
	Brinster, R.L. and Palmiter, R.D. 1984. Introduction of genes into the germ line of animals. Harvey Lect. 80:1‐38.
	Brinster, R.L., Chen, H.Y., Trumbauer, M.E., and Avarbock, M.R. 1980. Translation of globin messenger RNA by the mouse ovum. Nature 283:499‐501.
	Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K., and Palmiter, R.D. 1985. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. U.S.A. 82:4438‐4442.
	Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 21:2.9.1‐2.9.15.
	Capecchi, M.R. 1989. Altering the genome by homologous recombination. Science 244:1288‐1292.
	Chatot, C.L., Lewis, J.L., Torres, I., and Ziomek, C.A. 1990. Development of 1‐cell embryos from different strains of mice in CZB medium. Biol. Reprod. 42:432‐440.
	Clark, A.J., Harold, G., and Yull, F.E. 1997. Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice. Nucleic Acids Res. 25:1009‐1014.
	Erbach, G.T., Lawitts, J.A., Papaioannou, V.E., and Biggers, J.D. 1994. Differential growth of the mouse preimplantation embryo in chemically defined media. Biol. Reprod. 50:1027‐1033.
	Everett, J.W. 1969. Neuroendocrine aspects of mammalian reproduction. Annu. Rev. Physiol. 31:383‐416.
	Finney, M. 2000. Pulsed‐field gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5B.1‐2.5B.9.
	Gong, S., Zheng, C., Doughty, M.L., Losos, K., Didkovsky, N., Schambra, U.B., Nowak, N.J., Joyner, A., Leblanc, G., Hatten, M.E., and Heintz, N. 2003. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917‐925.
	Gordon, J.W. and Ruddle, F.H. 1981. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214:1244‐1246.
	Han, S.J., O'Malley, B.W., and DeMayo, F.J. 2009. An estrogen receptor alpha activity indicator model in mice. Genesis 47:815‐824.
	Heintz, N. 2000. Analysis of mammalian central nervous system gene expression and function using bacterial artificial chromosome‐mediated transgenesis. Hum. Mol. Genet. 9:937‐943.
	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.
	Lewandoski, M. 2001. Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2:743‐755.
	Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, New York.
	Psychoyos, A. 1966. Study of the relations of the ovum and ondometrium during delay of nidation or the first phases of the nidation process in the female rat. C. R. Acad. Sci. Hebd. Seances Acad. Sci. D 263:1755‐1758.
	Quinn, P., Barros, C., and Whittingham, D.G. 1982. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fertil. 66:161‐168.
	Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
	Vunder, P.A. 1970. Hormonal regulation of the process of ovum implantation. Usp. Sovrem. Biol. 70:106‐119.
	Whittingham, D.G. 1971. Culture of mouse ova. J. Reprod. Fertil. Suppl. 14:7‐21.
	Yoshinaga, K. 1988. Uterine receptivity for blastocyst implantation. Ann. N.Y. Acad. Sci. 541:424‐431.
	Zhao, S. 2001. A comprehensive BAC resource. Nucleic Acids Res. 29:141‐143.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Genetically Engineered Mice by Pronuclear DNA Microinjection

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation of Transgene DNA

Basic Protocol 2: Embryo Collection

Basic Protocol 3: Microinjection

Basic Protocol 4: Embryo Transfer

Support Protocol 1: Embryo Culture

Basic Protocol 5: Identification of Transgenic Mice

Figures

Videos

Literature Cited