Applications

The technique of introducing new genetic material into the germline of mammals has been a major development in biotechnology over the last decades. ”Transgenic animals“ are animals whose chromosomes contain stable, integrated copies of exogenous genes, additional copies of endogenous genes or gene constructs. They are frequently created by two different tech-niques: microinjection of DNA into the pronucleus of zygotes and injection of embryonic stem cells into blasto-cysts. In this Application Note, these techniques, as well as another technique for creating chimeras, the produc-tion of tetraploid mouse embryos, are discussed.

Note 011 | June 2006

Sonja Voss, Eppendorf AG, Hamburg, Germany

Transgenic animals

Introduction

Abstract

Although several steps in the evolution of transgenic technology had been performed in the 1970’s, transgen-esis was not widely recognized before Palmiter et al. [1] introduced the human growth hormone gene into mouse zygotes by pronuclear microinjection and transgenic off-spring demonstrated dramatic growth. Since then trans-genic techniques have been of immense importance, as they allow new approaches to life science reasearch that

general cell culture techniques cannot deliver [2]. The pro-duction of large quantities of complex, bioactive proteins like hormones or growth factors for therapeutic purposes (pharming) [3] is only one example of a wide range of dif-ferent applications for the study of genetic regulation, ani-mal development or disease pathology. For this purpose, foreign DNA is introduced into fertilized oocytes or em- bryos (blastocysts) of mice, rats and other mammals [1,4,5].

Application Note 011 | page 2

subsequently transferred into the uterus of pseudopregnant recipient animals. The offspring is screened (e.g., by PCR with Eppendorf Mastercycler ep) to confirm a successful integration of the gene of interest for use in further studies. Eppendorf workstation setupEppendorf offers the complete equipment for an optimal workstation:2 x TransferMan NK 2 micromanipulator1 x FemtoJet or FemtoJet express microinjectorFemtotip II microcapillariesMicroloader1 x CellTram Air microinjectorVacuTip microcapillaries One TransferMan NK 2 micromanipulator is used for mov-ing the holding capillary and the other is used for moving the injection capillary (Fig. 4). To hold the embryo, a CellTram Air microinjector in combi-nation with a VacuTip microcapillary is used.For injecting the DNA, a FemtoJet or a FemtoJet express is used in combination with the microinjection capillar-ies Femtotip II. Both models of FemtoJet are ideal for the injection of DNA and deliver reproducible results. The choice of model depends on pressure and troughput requirements. Sharing the same operation concept, the FemtoJet has a built-in compressor and works as a stand- alone unit, while the FemtoJet express uses pressure from an external supply. The injection angle should be as flat as possible: microcap-illaries for the pronuclear injection, for example Femtotip II, are usually not bent; therefore we recommend also using a 10° - adapter (with the A-Head with TransferMan NK 2 (Fig. 5)).

The pronuclear microinjection method of producing a trans-genic animal is based on the introduction of linear DNA sequences into the chromosomes of fertilized eggs. The foreign DNA must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage in order for the animal to be born with a copy of this new information in every cell.For several hours following the entry of the sperm into the oocyte, the male and the female pronuclei can still be seen individually under a normal light microscope and they have not fused yet into a so-called zygote. The foreign DNA may be injected into either pronuclei with no difference in results (Fig. 3); however, the DNA is typi-cally injected into the male pronucleus because it is slightly larger and closer to the oocyte surface. These oocytes are

Fig. 2: Microinjection techniques

Microinjection of linear DNAinto the pronucleus

Mouse Transfected ES cells

Fertilized egg(one cell embryo,pronuclear stage)

Blastocysts

Microinject gene of interestas linear DNA into

male or female nucleus

ES cells injected intocavity of blastocysts

Introduce several such embryos into

pseudopregnant mouse

Birth

Transgenic linehas to be bred

Introduce several such blastocysts into pseudopregnant mouse

Chimeric linehas to be bred

Birth

EmbryoInjected cells become incorporated in inner cell massof host blastocyst

Transfer of embryonic stem cells (ES cells) into blastocysts

1

2

3

4

5

6

7

Fig. 3: Microinjection of DNA into the pronucleus of a fertilized oocyte

Microinjection of DNA into the pronucleus

Microinjection techniques

tion or a shutdown in the embryonic development at later stages. There are different methods for purifying the DNA before microinjection: classical methods, such as CsCl ethidium bromide gradient centrifugation for plasmid DNA preparation or the extraction of DNA with organic solvents followed by several purification steps are often very time-consuming. Alternatively, DNA purification kits provide DNA of excellent quality and purity for use in microinjection.Another important factor for success is the concentration of DNA which directly affects efficiency. Low concentrations of DNA may result in a low rate of integration, whereas high concentrations can be toxic for the zygote. DNA concentra-tions for pronuclear injections usually range between 1.5 and 2 ng/µl [6].The preparation of the injection buffer should be handled with care, as the diameter of capillaries is very small and particles in the injection solution can quickly result in block-age of the capillary. TE buffers with an EDTA concentra-tion of 0.1-0.3 mM are usually used for DNA injection into mouse embryos [6]. If problems with blocked capillaries occur, the DNA solution can be diluted or additionally fil-tered through a 0.22-µm filter (for constructs smaller than 5 kb only).

Preparation of equipmentThere are different injection chambers (plastic Petri dish with glass bottom, depression slide injection chamber or metal frame/glass slide injection chamber [6]) available to serve as a microenvironment for the zygote during the injection pro-cess. One drop of M2 medium is placed in the chamber. The zygotes to be injected (10 to 20) are placed in one area of this drop. The whole droplet is covered with oil. FemtoJet and FemtoJet express offer an automatic (injec-tion time is preset) and a manual injection mode. The injec-tion is triggered by a hand control or optional foot control. For pronuclear injection we recommend using the manual mode because the pronuclei differ in size and the pronu-clear swelling can be achieved for each individual oocyte. Depending on the inner diameter of the injection capillaries, the basic settings for the compensation pressure (pc) and the injection pressure (pi) must be determined empirically. When using Femtotip II, start with the following settings: pc = 15-20 hPa, pi = 80-120 hPa Both VacuTip (holding) and FemtoTip II (injection) pipettes must be fitted into the capillary holder and are carefully aligned before injection.The Microloader, a very long and fine pipette tip is used to load the Femtotip II microcapillary with DNA from behind.

When this setup is used in combination with an inverted microscope that is equipped with a Hoffman Modulation Contrast or a Differential Interference Contrast, microinjec-tion can be performed quickly and simply.

Application Note 011 | page 3

Fig. 4: Workstation setup for microinjection of DNA into the pronucleus: TransferMan NK 2, CellTram Air and FemtoJet in combination with a Leica DMI 6000 microscope

Fig. 5: 10°- adapter with A-Head

Materials and methods Many different organisms may be used for transgenic experiments. Although the general procedure is the same, this Application Note focuses on the manipulation of the mouse embryo.

Purification and preparation of DNA for microinjectionSuccessful results depend heavily on the purity of the DNA. The slightest amount of contaminating agents will harm the oocyte and result in either a lysis shortly after microinjec-

Application Note 011 | page 4

Operation of the micromanipulator TransferMan NK 2 Operating the TransferMan NK 2 micromanipulator, with its few self-explanatory keys, is extremely simple: a joystick provides easy movement of the capillary in any direction (x, y, z), and each TransferMan NK 2 can store up to three independent positions in memory; by simply pressing the joystick button twice, the capillary can be moved to one of the three preprogrammed positions.

Position A

Object slide with droplet

Position B

Hold Inject

Position 2

Position 3

Position 1

Fig. 6: Storage of positions for microinjection of DNA into the pronucleus

On the holding side Position 1 is stored when the capillary has reached the area where the uninjected zygotes are placed. The CellTram Air microinjector and VacuTip holding capillaries can easily take up a single zygote, which is then moved to a central position for injection of the DNA. This position is stored as Position 2. After injection, the zygote is transferred to another area of the dish to keep the unin-jected zygotes separate from the injected ones. This area is set as Position 3.On the injection side, Position B brings the injection capil-lary in focus after fixing a zygote on the holding side and Position A serves as the “parking position“. MicroinjectionA zygote in the pronuclear stage is brought into the injec-tion position. By using the CellTram Air microinjector, the zygote is firmly held by the VacuTip holding pipette. The focus is set to the midplane of the pronucleus, and, if necessary, the zygote can be repositioned. The tip of the injection capillary is aligned to the same focal plane and is pushed through the zona pellucida and the cytoplasm until it enters the pronucleus. Approximately 1–2 pl DNA solution is injected during every injection process, and the injection is considered successful if the pronucleus increases in size.

If injection accidentally occurs into the cytoplasm then the size of the pronucleus does not increase and the injection drop shows a different contrast.If the tip comes too close to the endogenous DNA during the injection process, the DNA might stick to the capillary when it is pulled out; if this occurs, the zygote should be discarded.

Fig. 7: ES cell transfer into blastocyst

The first step is the removal of ES cells from a blastocyst. After transfection of the ES cells, selection, cloning and screening methods make it possible to detect ES cell clones that demonstrate the desired, site-specific recom-bination. After microinjection of the genetically modified ES cells into blastocyst-stage embryos the ES cells divide and become part of the embryo.The following chimeric animals will subsequently transmit the recombinant genotype to their offspring, if the ES cells have contributed to their germ cells.

Embryonic stem cells (ES cells) are derived from the inner cell mass of blastocysts. These cells are pluripotent, which means that they can develop into almost any type of tis-sue. ES cells are used for more precise modifications of the mouse genome. This technique makes it possible to insert as well as to modify DNA sequences. Knock-out, knock-in and conditional mutant mice [7] can be produced with this method.

Injection of ES cells into blastocyst-stage embryos

Holding capillary VacuTip

Transfer capillary TransferTip (ES)

Blastocyst

Application Note 011 | page 5

Eppendorf workstation setup2 x TransferMan NK 2 micromanipulator1 x CellTram vario microinjectorTransferTip (ES) microcapillaries1 x CellTram Air microinjectorVacuTips microcapillaries

Fig. 8: Workstation setup for ES cell transfer into blastocysts: TransferMan NK 2, CellTram Air and CellTram vario in combina-tion with a Olympus IX 70 microscope

For the transfer of ES cells into blastocysts, two TransferMan NK 2 devices (Fig. 8) are used for controlling the holding and transfer capillary. CellTram Air used in combination with a VacuTip actually holds the blastocysts.The CellTram vario used in combination with a TransferTip (ES) transfers the ES cells. As previously mentioned, one inverted microscope with Hoffman Modulation Contrast or Differential Interference Contrast makes the ES cell transfer quick and simple. Materials and methods Many different organisms can be used for transgenic experiments. Although the general procedure is the same, this Application Note focuses on the manipulation of the mouse embryo.

Electroporation of ES cellsThe most commonly used method of modifying the genome of ES cells is by introducing a DNA fragment through electroporation. The Eppendorf Multiporator® is very well suited for this kind of application. It’s Soft PulseTM technology applies extremely short electric pulses to provide the highest survival rates - cell-damaging influ-ences, such as changes in pH values, aluminum release and electrophoresis of the cell contents, are minimized. Voltage and pulse duration can be set directly and its pat-ented electronic pulse discharge precisely maintains these parameters independent of the sample resistance to ensure reliable and reproducible results.Eppendorf’s specially designed electroporation buffers complete this optimally balanced system for efficient and gentle electroporation of eukaryotic cells; however, it is important to note that the electroporation conditions must be optimized for each DNA construct. Detailed protocols are available on “www.eppendorf.com”.

Preparing the blastocyst for injectionBlastocysts for injection should be of highest quality, otherwise the injection process can become quite difficult or the developmental potential can be limited. The timing should be well calculated as well: only well-expanded, but not overexpanded or hatched embryos should be used. The embryos should be handled with great care during the whole experiment. Preparing ES cells for injection ES cells for injection should show an ideal, undifferentiated morphology in culture. Growth should be exponential, and the cells should be harvested at subconfluent density.The ES cells are prepared one hour prior to starting the actual injection procedure. It is important to keep the feeder-dependent ES cells growing on good-quality mouse embryonic fibroblasts (MEF) at all times to minimize the risk of differentiation. For injection, MEF can be disturbing and should be removed [6]. Preparation of technical equipmentBoth the VacuTip holding and TransferTip (ES) injection pipettes are fitted into the capillary holder and care-fully aligned. The preparation of the injection chamber is described above. Additionally, a few hundred ES cells are introduced into the injection chamber.

Application Note 011 | page 6

Operation of the micromanipulator TransferMan NK 2 As previously mentioned three independent positions can be defined and stored for each TransferMan NK 2.

Position APosition B

Inject

Object slide with droplet

Hold

Position 3

Position 1

Position 2

Fig. 9: Storage of positions for ES cell transfer into blastocysts

On the holding side, Position 1 is stored when the capillary has reached the area where the uninjected embryos are placed. The CellTram Air microinjector and VacuTip holding capillary can easily take up a single embryo, which is then moved to a central position for injection of the ES cells. This position is stored as Position 2. After injection, the embryo is transferred to another area of the dish to keep the uninjected embryos separate from the injected ones. This area is set as Position 3. Injection of ES cells Under high magnification (40 x), individual ES cells can be selected according to size and shape. Fifteen to twenty ES cells are taken up into the capillary with a minimal amount of medium and are positioned near the tip.An embryo in the blastocyst stage is brought into the injection position (Fig. 9, Position 2). With the CellTram Air microinjector, the embryo is firmly hold to the holding pipette. After positioning the blastocysts (the inner cell mass must be at either the 6 o’clock or 12 o’clock position) the tip of the injection needle is aligned to the same focal plane as the equator of the blastocyst; carefully touch the surface of the embryo with the tip to find the right plane.With a single, continous movement the loaded injection capillary is pushed into the blastcyst cavity and the cells

are slowly expelled into the cavity. It is critical that no oil bubbles or lysed cells are inserted into the blastocyst. After injection the capillary is slowly drawn out of the embryo. When the pipette is completely removed, the blastocyst will collapse.

Fig. 10: Two-cell embryos before fusion

Combining tetraploid blastocysts with either embryonic stem (ES) cells or diploid embryos has become an estab-lished technique for creating mouse chimeras, e.g., by gene targeting. It thus enables fast and efficient analysis of gene function. In both methods the tetraploid cells are not able to con-tribute to the embryo itself, but instead create the primitive endoderm derivatives and the trophectoderm [8]. The cre-ation of an ES cell tetraploid embryo chimera, in particular, provides an important research tool. Due to cell comple-mentation, complete segregation of descendants of ES cells and tetraploid cells is achieved, resulting in fetuses and viable offspring that are completely derived from ES cells [9,10,11].ES cell tetraploid embryo chimeras are employed in several approaches: they enable rapid analysis of mutant phenotypes that are derived from genetically modified ES cells, elimi-nating time consuming breeding and germ line transmission.

Production of tetraploid mouse embryos

Application Note 011 | page 7

Fig. 11: Multiporator with Micro fusion chamber for observing electrofusion under the microscope

The tetraploid blastocysts are produced by electrofusion using the Eppendorf Multiporator with fusion module. The fusion module brings cells into contact with one another in an electrical alternating-current environment and then fuses them via a direct-current pulse. For a detailed protocol please refer to Eppendorf Application Note No. 116: “Production of tetraploid mouse embryos by electrofusion” by Ronald Naumann [16].

Results

Both microinjection of DNA into the pronucleus and ES cell transfer into blastocysts can easily be performed with the Eppendorf TransferMan NK2 micromanipulator, CellTram Air, CellTram vario and the FemtoJet or FemtoJet express. Simplified automation of repetitive tasks allows users to concentrate on the micromanipulation procedure. In com-bination with Eppendorf’s high precision microcapillaries VacuTip, FemtoTip and TransferTip (ES) these manipulation systems enable rapid work with consistent results [14, 15]. The Eppendorf Multiporator is an ideal device for the pro-duction of tetraploid mouse embryos by electrofusion [16].

References

[1] Palmiter R.D., Brinster R.L., Hammer R.E., Trumbauer M.E., Rosenfeld M.G., Birnberg N.C., Evans R.M.: Dramatic growth of

mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. (1982) Nature 300, 611-615

[2] Chan AWS: Transgenic animals: Current and alternative strategies. (1999) Cloning, 1:25-46.

[3] Wall, R.J., Kerr, D.E., and Bondioli, K.R. : Transgenic Dairy Cattle: Genetic Engineering on a Large Scale. (1997) J.Diary Sci. 80,2213-2224

[4] Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., Ruddle, F.H.:

Genetic transformation of mouse embryo by microinjection of purified DNA. (1980) Proc.Natl.Acad.Sci USA 77, 7380-7384

[5] Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL: Production of transgenic rab

bits, sheep and pigs by microinjection. (1985) Nature 315 (6021), 680-3.

[6] Nagy,A., Gerstenstein, M., Vintersten, K., Behringer, B.: Manipulating the Mouse Embryo, A Laboratory Manual. Third edition

(2003) Cold Spring Habor Laboratory Press

[7] Barbinet, C.:Transgenic Mice: An irreplaceable tool for the study of mamalien developmental biology. (2000) J.Am.Soc.Nephrol 11:88-94

[8] Tarkowski, A. K., Witkowska, A. and Opas, J.: Development of cytochalasin Betainduced tetraploid and diploid-tetraploid

mosaic mouse embryos. (1977). J. Embryol. Exp. Morphol.41: 47-64.

[9] Nagy, A., Gocza, E, Diaz, E. M., Prideaux V. R., Ivanyi, E. Markkula, M. and Rossant. J.: Embryonic stem cells alone are able

to support fetal development in the mouse. (1990). Development 110: 815–821.

[10] Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J.C.: Derivation of completely cell culture-derived mice

from early-passage embryonic stem cells. (1993). Proc. Natl. Acad. Sci. USA 90 (18): 9424-9428.

Gene functions in the embryonic versus extra-embryonic lineages can be tested, and complex phenotypes can be produced by generating embryos that carry mutations of multiple genes [12]. This method is also the only way to analyze genes known to be lethal to heterozygous embryos [13].

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Article Description Order no. international

Order no. North America

TransferMan®NK2 Proportional micromanipulator for suspension cells and microdis-section

5188 000.012 920000011

CellTram® Air Manual pressure device for holding suspension cells, complete with pressure tube and universal capillary holder with grip head

5176 000.017 920002021

CellTram® vario Hydraulic manual microinjector with gear, complete with pressure tube, filling tube, syringe, oil and universal capillary holder with grip head

5176 000.033 920002111

FemtoJet® Programmable microinjector with integrated pressure supply 5247 000.013 920010504

FemtoJet® express Programmable microinjector with external pressure supply 5248 000.050 920010521

VacuTip Holding capillary, sterilized, tip angle 35° inner diameter 15 µm, outer diameter 100 µm

5175 108.000 930001015

TransferTip® (ES) Transfer capillary, sterilized, tip angle 20°, inner diameter 15 µm, outer diameter 20 µm

5175 107.004 930001040

Femtotip II Microinjection capillary, inner diameter 0.5 µm, outer diameter 0.7 µm

5242 957.000 930000035

Microloader Special pipette tips for filling Femtotips from the rear 5242 956.003 930001007

Multiporator® For eucaryotic cells and cell fusion 4308 000.031 940000700

Micro fusion chamber Gap width 0.5 mm 4308 031.000 940001234

Application Note 011

Ordering information

References[11] Eggan, K., Akutsu H., Loring J., Jackson-Grusby L., Klemm M.,. Rideout W. M III, Yanagimachi R. and Jaenisch R.: Hybrid

vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. (2001) Proc.

Natl. Acad. Sci. USA 98: 6209–6214

[12] Tam, P. P. L. and Rossant, J.: Mouse embryonic chimeras: tools for studying mammalian development. (2003) Development 130: 6155-6163.

[13] Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K.,

Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W. and Nagy, A.: Abnormal blood vessel development

and lethality in embryos lacking a single VEGF allele. (1996) Nature 380: 435-439.

[14] Peloquin, J.J., Thibault, S.T., Schouest, L.P., Miller, Jr., Miller, T.A.: Electromechanical Microinjection of Pink Bollworm (Pecti

nophora gossypiella) Embryos Increases Survival. (1997) BioTechniques 22: 496-499

[15] Al-Hasani, S., Ludwig, M., Karabulut, O., Al-Dimassi, F., Bauer, O., Sturm, R., Kahle, D., Diedrich, K.: Results of intracytoplasmatic sperm

injection using the microprocessor controlled TransferMan Eppendorf manipulator system. (1999) MEFS Journal Vol. 4, No.1, 41-44

[16] Eppendorf Application Note 116 (Tetraploid embryos) , www.eppendorf.com

We thank Ronald Naumann, Transgenic Core Facility, MPI of Molecular Cell Biology and Genetics, Dresden, Germany for providing us with pictures (Fig. 3,7,10)