Experiments on Prokaryotic Microinjection Technique

Summary

A technique for preparing transgenic mice by prokaryotic microinjection is described. Prokaryotic microinjection, which involves the injection of DNA into haploid prokaryotic fertilized eggs, is a widely accepted technique. Transgenic mice can be constructed from simple promoters/cDNAs or from large vectors containing genomic fragments such as mucoids, yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC).

Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".

Operation method

Preparation of microinjection chambers and microtools

Move

Preparation of microinjection chamber and microtools Materials

reagents

Anhydrous ethanol (95 % and 70 %)

Hydrochloric acid (1%)

SafetyCoat Nontoxic Coating (JTBaker 4017-01) or Sigmacote (SigmaAldrich)

Instruments

4in blunt stainless steel needle (Custom order: Hamilton Co., Reno, Nevada) or plain 18G skin puncture needle attached to a piece of 1/32 (0.8in) I.D. flexible tubing (Saint-Gobain PerformancePlastics AJC40001) with a fixation needle mounted on the back of the tubing. The needle is attached to the back of the hose.

Diamond or Diamond Pen

DIC-compliant optical slides (2 5 mmX 75 mm, pre-cleaned)

Vacuum sanding oil (Dow Corning 1597418)

Miniature Bunsen burner

Replace the burner with an obtuse 18 G needle and adjust the Bunsen burner to emit a very small flame.

Micro Capillary (100ul )

Using a diamond or diamond pen, cut the capillary tube into 2 2mm lengths (two sections per microinjection chamber).

Microforging instrument (DeFonbrune^type or Narishige-type) Compound microscope with binoculars; 10 X optical eyepieces, one of which is fitted with a cross micrometer; objective lenses 4X, IOX and 20X (long working distance)

P-87/P-97 Flaming-Brown with boxed drill wire (2.5 mmX2.5 mm or 3 mmX3 mm).

Microelectrode horizontal needle puller (Sutter Instruments, Novato California)

Plastic spherical pipettes (optional)

Slide cassettes for dust-free storage

Silicone (GE Silicone II), vacuum silicone oil, or wax for attaching capillary tubes to the slides.

Thick-walled capillary tubes (I.Omm OD X 0.58 mm ID) for needle fixation (World Precision Instruments, Sutter Instruments)

Thin-walled single-tube filament capillary for capillary needles (I.Omm OD X 0 -75 mm ID) (World Precision Instruments, Sarasota, Florida; Sutter Instruments)

Syringes are used to generate positive pressure on the needle during silicification (optional, see step 13).

18G dermal puncture needles (1/32[100])<3/32[OD]\1/32[wall]) (Saint-Gobain Performance Plastics AJC40001) with 6 to 8 cm long obtuse hose attached to a 20 cc disposable syringe with a snap-on rubber cap.

METHODS _

Preparation of microinjection chamber

1- Adhere a 22m m length of IOOul capillary tube to a 25m m X 75m m pre-prepared slide with a small amount of silica gel (GESiliconeII), with the capillary tube parallel to the long side of the slide and 2 m m from the edge.

2 - Glue another 22m m long capillary along the edge at the same position on the other side of the slide and dry the glue overnight in a running tissue culture incubator.

3 - Thoroughly wash the slide with 1 % hydrochloric acid, wash it several times with evaporated water and finally with ethanol.

4- Add silica gel solution between slides with a plastic spherical pipette that can be used freely, and then rinse several times with strong-flow distilled water.

SafetyCoat Nontoxic Coating is the best, but S gmacote can also be used.

5- Dry the microinjection slides in a tissue incubator and place them in a plastic slide box for dust protection.
Microtool making: pulling the embryo fixation needle

6- Heat a 5mm long section of thick-walled capillary tube (I.0 m m OD X O.58 m m ID ) at 2/3 from the end, and lengthen it to 30 m m , so that the diameter of the tube is between 100 ~ 150 um.

7. Cut the elongated tube with a diamond pen at a distance of 10-15mm from the thinning point of the longer end of the fixed needle, and break between the long part and the short part.

8. Check the fixation needles on the microforator to make sure that the needles are symmetrical and have no tips or cracks, and to make sure that the fixation needles are ready to fix the embryo in the correct position during microinjection. If necessary, cut the pipette with a thick wire on the microforator to create a symmetrical surface.

9. Smooth the pipette with the coarse wire of the microforger and make the opening of the pipette as thin as 15 to 20 um. If necessary, make a 5° bend in the projection of the pipette so that the fixation needle can enter the microinjection drop parallel to the face of the injection chamber.

10. Flatten the thick end of the fixation needle with the flame of a miniature Bunsen burner, but be careful not to close the opening.

Microtool making: pulling a microinjection needle

1 1 . A microinjection needle is created with the Suttel Institutes P-97 Microelectrode Horizontal Needle Puller using the system's default parameters: Heat = Ram PvAlue; Pull = 50; Delay = 80; Pressure = 200. Adjusting the parameters creates a gradually tapering needle of 7-9 m m in overall length, allowing the needle to change rapidly from a barrel of approximately 2(Vm) to the last 0.5 m m. The needle is then pulled to the last 0.5 m m. The needle is then pulled to the last 0.5 m m. The parameters were adjusted to make a needle of 7 to 9 m m overall length tapering rapidly from about 2(V m) to the final 0.5 m m.

12- Gently sharpen the tip of the needle on the glass bead of the microtome so that the pipette tip is at 0.2 to 0.5 um (Mann and McMahon 1993).

13. Optional step: Siliconize the microinjection needle before sharpening the pipette tip to help reduce the adhesion of cellular material to the tip and increase the number of eggs injected with a single needle. The procedure is as follows:

a- Attach the needle to a 2 0 c c syringe attached to an IO c m length of I D 1 / 3 2 hose.

b - Apply positive pressure to the syringe and immerse the tip of the needle in SafetyCoat Nontoxic, diluted with 95 % ethanol, twice for 5 s each time.

c. Immediately rinse the needle tip with 9 5 % ethanol, followed by 70 % ethanol, and then immerse in 0. 2 2 um sterile filtered distilled water.

d. Dry the tip of the needle and then sharpen it on the microforger as described in step 12.

Angle adjustment for fixed and microinjection needles

14. When using the Leica-type micromanipulator, it is not necessary to make further angular adjustments after pulling the microinjection needle and the fixation needle. When using a Rail-Mounted Narishige-type micromanipulator, it is necessary to bend the tips of the micromanipulator needle and the fixation needle by 30° to 35° so that the tip of the needle can enter the microinjection drop parallel to the microinjection chamber.

Preparing DNA for Microinjection material

Reagents

Agarose gel

Ammonium acetate (5m ol/L )

DNA microinjection buffer (100 m m o l/L T ris-H C 1 p H 7.5, 0-lm m o l/L ED T A, injection water to meet all U SP M o n o g ra p h requirements [W F I; Invitrogen])

Filter using 0-22um SFCA sterile filters (Millipore).

Ethanol (100 % and 95 %)

Phenol: chloroform

Plasmid

Restriction endonucleases for digestion of plasmids

Sucrose solution

1 0 % (m/V ) and 40 % (to/V ) in lm ol/L NaCl, 10 mmol/L Tris-HCl pH 8. 0, lm m ol/L EDTA prepared in W F I water. Filtering was performed using a 0.22 fzm SFCA sterile filter (Millipore).

T E buffer (10 m m o l/L T ris-Cl p H 8. 0, lm m o l/L ED T A )

Instrumentation

Centricon-IOO micro-concentrator (A m e rsh a m )

Gradient Forming Instrument (e.g., S G 30 g ra d ie n t fo rm e r; A m e rsh a m )

Low-flow worms (〇 -0.3 ~ 8.2 m l/m in ) (Y W R S cien tific)

Microcentrifuge (I.7m l, sterile)

If necessary, remove dust particles by filtering endotoxin-free distilled water through a sterile 0.22 um filter.

Needle (25 G )

Rubber Plug

The rubber stopper is to have an 18 G skin puncture needle passing through the center of the stopper from top to bottom and to fit nicely in the upper part of the ultracentrifuge.

Ultracentrifuge tubes (B eckm an U ltra -C le a r 344060)

Ultracentrifuge and tumbling-type ultracentrifuge rotors (e.g., S W 40 Ti ; B eckm an-C o u lte r)

Methods

1- Cut 50ug of plasmid with restriction endonuclease to release the transgene insert fragment from the plasmid. Mucilage DNA was linearized with restriction endonuclease prior to purification.

2- Extraction with phenol: chloroform, followed by precipitation with 1x volume of 5m o l / L amine acetate, 2.5x volume of 1,000 % ethanol, centrifugation at 13 OOO g for more than 30m i n , and suspension in 50 ug T E buffer.

3- Pour two linear 1 0 % ~4 0 % density gradients of sucrose in ultracentrifuge tubes according to the instructions for the density gradient formers, for a total volume of approximately 14 ml.

4- Add the cut DNA to one density gradient centrifuge tube and use the other density gradient tube to separate the other fragments or as an equilibrium.

5- Precisely equilibrate the sucrose density gradient tubes with microinjection buffer and centrifuge the plasmid cut fragments at 4°C, 35 OOOr/min for 4 h; centrifuge the viscous pellet at 35,000r/min for 24 h. The plasmid cut fragments are then centrifuged at 4°C, 35 OOOr/min for 4 h.

6 . When the centrifugation speed of the ultracentrifuge begins to decrease place 80 sterilized, sterile I.7 m l microcentrifuge tubes on a rack, insert a rubber stopper/pin in the upper part of the ultracentrifuge tube, and remove approximately 2 m l of the liquid containing the DNA from the upper part of the sucrose density gradient.

7- Place your finger in the open position on the stopper and push the 25 G needle into the bottom of the ultracentrifuge tube, with the 25 G needle going into the density gradient liquid less than 5 mm. control the flow rate with the needle on the stopper, and collect approximately 0.2m l of small fragments in each collection tube.

8. Take a 5ul aliquot of agarose gel for every 4 collection tubes to confirm that the first and last collection tubes contain only the transgenic insert fragments. Repeat the gel analysis for all collection tubes, adding vector+insertion fragment and insertion fragment to the spot wells on both sides of the gel, respectively.

9- Combine all collection tubes containing only the transgene insert fragments and perform 5 to 7 consecutive buffer changes using a Centricon-IOO microconcentrator with microinjection buffer. For plasmids, the yield is 25% to 50% of the total DNA at the start, and for mucilages it is 10% to 25%.

10 . The concentration of DNA is accurately measured spectrophotometrically, and small samples are analyzed on agarose gels for comparison with standards of similar size.

11. Store concentrated DNA at 4°C and dilute DNA to 2 to 3ug/ml with microinjection buffer. Prepare several 1004 aliquots of DNA for microinjection in sterilized, dust-free microcentrifuge tubes. Store at 4°C for up to one week. Spare DNA can be frozen at 1 20°C, but be careful to suspend the DNA completely when diluting for microinjection.

Supernumerary Ovulation and Embryo Collection Materials

reagents

IV-S type bovine testicular translucency enzyme (l0 m g /m l; S igm a-A ld ric h ) in M 2 (storage solution 4 0 X )

Store aliquots at 20°C. Dilute to 250ug/ml with M2 before use.

CO2 for euthanasia of embryonic donor mice (optional, see step 4b)

Embryonic donor mice

Human chorionic gonadotropin (H G G ) (Sigma-Aldrich)

Dilute to 500 IU/m l in sterile 0-9 % saline and store 0-I m l aliquots at 80°C for up to 6 months.

Light mineral oil (Sigma-Aldrich, for paired tests of toxicity or embryo testing).
Store 30 to 40 ml of mineral oil in a loosely capped bottle in a swim chamber, and using this "balance," the mineral oil goes on to cover the CZB.

(i) Culture solution.

Culture medium

C Z B medium: glucose-free sodium bicarbonate buffered embryo culture medium for all single-cell embryo cultures, regardless of strain (ChatotetaL 1989). Storage solution is prepared with salt and sterilized endotoxin-free H2O (Nagyetal.2003).

M 2 Medium: H E P E S ■ Buffered embryo suspension medium (Quin n etal. 1982). Prepared in sterilized endotoxin-free evaporated water (Nagy et al. 2003) or purchased (Sigma-Aldrich of Specialty Media/Chemicon, Temecula, California). Other mouse embryo culture media (KSOM-AA, M16) can be purchased (Specialty MEDIA/Chemicon, Temecula, California).
Specialty Media/Chemicon)".

Pregnant horse serum gonadotropin (PMSG) (e.g., Sigma-Aldrich or the National HormonePeptide Program [Dr. A.F. Parlow, Harbor UCLA Medical Center, Los Angeles, California]) "

Prepare a 500 IU/m l storage solution in 0.9 % sterilized saline and store the sterilized 0.ImI aliquot at 80°C for up to 6 months.

Saline (0.9 %, sterilized) is used as an injection solution. Saline (0.9 %, sterilized) is used as a storage solution to dilute the hormone prior to injection.

Apparatus

Float tank, pre-conditioned to 37°C (humidified, 5 % CO2)

The mouth pipette was used for embryo fixation

A 16- to 18-inch piece of rubber tubing (1/8X3/16X 1/32) with a 10(^1) capillary attached to one end is inserted into the other end of the rubber tubing by cutting off 2 to 3 mm from the tip of the attached isolation plug pipette tip. The large end of the Pasteur pipette, used as an embryo fixation needle, is placed into the large attached isolation plug pipette tip, and the other end is a mouth pipette (HPIHospital Products, Apopka, Florida). isolation plug pipette tip and the other end was a mouth pipette (HPIHospital Products, Apopka, Florida).
Petri dishes (3 cm)

Stereo zoom microtomes (e.g. Leica MZ7.5) that illuminate the embryo while moving it between different cultures.

Clock forceps (Cumont no. 5 ) (two pairs), fine dissection scissors (one pair) (World Precision Instruments; Roboz Surgical Instrument Co., Inc., Gaithersburg, Maryland)

Methods

Supernumerary ovulation in embryo donor mice

1. Generally used in prokaryotic microinjection experiments in female mice from mouse strains that ovulate in excess of 3 to 5 weeks of age before the onset of the estrous cycle of origin, and some strains of mice are more effective in responding to hormones after the onset of the estrous cycle of origin.

2. The standard dose of PMSG is 5I U/mouse, administered by intraperitoneal injection 6-IO h after the start of the photoperiod (e.g., 12:00 PM to 4:00 PM with lights on at 6:00 PM). For some strains of mice increasing or decreasing by 2.5IU/mouse will work better.

3- Stimulate ovulation by intraperitoneal injection of HGG at a dose of 5 IU/mouse 46-48 h after PMSG injection. The dose of HGG generally matches the amount of PMSG injected earlier. Embryo donor female mice are paired with fertile male mice immediately after HCG injection. Female mice from self-incompatible lines (e.g., C57BL/6J) have an increased yield of normally fertilized eggs when they are not paired with males 4 to 6 h after HCG injection. Detection of intravaginal mating plugs in supernumerary ovulating females is the strongest evidence that mating has occurred.

4. Recovery and maintenance of fertilized single-cell embryos are described as follows

a. Prepare four 3 cm Picasso dishes, each containing five rows of five 10ul droplets of CZB embryo culture solution, labeled ("Washl", "pre", "Wash2", and "Injected"), covered with mineral oil, and equilibrated in a cell culture incubator curried with 95% CCV air. Allow the dishes to equilibrate in the tissue culture incubator for >3Omin before inoculating the embryos.

b. After an intermediate dark cycle of 10 to 12 h, euthanize embryonic donor mice by aspiration of CO2, or by cervical dislocation.

c- Dissect the oviduct of each mouse, being careful not to tear the uterine tibia containing the single-celled egg surrounded by accumulated material.

d- Rinse the oviduct wall of each mouse in H E P E S ■ buffered embryo suspension medium M2.

e. Prepare 0 ■ 5 m l of Embryo Treatment Medium containing approximately 250 ug/m l of Bovine Testis Hyaluronidase, Type IV-S.

f. Take a stack from the ovary of each mouse, place it in a drop of M2 medium containing hyaluronidase, and tear open the uterine tibia with clockwork forceps.

g. Stir the stack in the droplet containing hyaluronidase until the stack of cells disappears. Collect the oocytes and rinse immediately with M2 medium without hyaluronidase. Move quickly.

h. Rinse 40-60 sets of embryos in 6 drops in a "Washl" dish, then place a drop in a "Pre" dish and transfer the embryos to a CO2 incubator. Prokaryotes will become visible after an intermediate dark cycle of 10 to 14 h. Microinjections should be performed as soon as possible after detection of the prokaryote.

Microinjection material

reagents

DNA solution for microinjection (see protocol 2 for preparation)

Fertilized eggs for microinjection (see Scheme 3 for preparation)

Gas (Sigma-Aldrich)

Fluorine is used to add embryo fixation needles when used with oil plus a microinjector.

Vacuum sand oil (D o w C o m m i n g 1597418)

M 2 Foundation

Petri dishes containing drops of C Z B medium (W ash 2 and Injected dishes prepared in step 4a of Option 3)

Apparatus

Absorbent paper

Coverslip (22 mmX22 mmXlmm)

Embryo fixation pins (see protocol I for preparation)

Inverted microscopes (high quality) with 4X brightfield, IOX brightfield, 3O~40X D IC or HoffmanModulation contrast optics are used for microinjection (e.g. LeicaMicrosystems, Wexlar, Delaware; NiKon USA, Melville, New York; Olympus America Inc. York; Olympus America Inc., Melville, New Yord; Carl Zeiss Imaging Inc., Thornwood, New York).

A 2 6 mm X 7 6 mm X 1. 5 m m slot machined as the center of the microscope holder insert holds the microinjection chamber in precise linearity . The lid of a 6 0 mm or IOO mm Pi Petri dish can be used with a Hoffman modulated phase contrast optic.

A manual microinjector controls the inhalation of the embryo fixation needle (e.g. Eppendorf, Narishige).

Microinjection chamber (see protocol 1 for preparation)

Microinjection needles (see Option 1 for preparation)

Pair of micromanipulators (left and right)

Mechanical micromanipulators (Leitz) require a base plane with rails, and hydraulic micromanipulators (Narishige International, New York) require rails with a microscope on top.

Pasteur pipette (sterilized)

Pipette Rack/Tool Sleeve

Inflatable Shock Tube (Kinetic Systems Inc., Boston, Massachusetts)

Inflatable microinjection devices (e.g. Femtojit Eppendorf AG, Hamburg)

Used to remove the blister injector from the microinjection needle.

Blunt 18 G laparotomy needle with 6 to 8 cm long hose (1/32 ID X 3/32 OD X 1/32 wall) (Saint-Gobain Performance PlasticsAJC4 0 001) attached to a 20 cc syringe with a snap-on rubber cap for casual use.

UV Spectrophotometer

Methods

Construction of microinjection drops

1. If the 2ug/ml DNA solution prepared in Scheme 2 is pre-frozen, mix it thoroughly to ensure that the DNA is completely solubilized. Check the concentration of the solution with a UV spectrophotometer (A260).

2 . 4°C, 14 000 g-centrifuge DNA solution for 30 min on ice.

3. Fill the two spaces of the upper capillary of the microinjection chamber (see Scheme 1 for preparation) with real silicone oil, and aspirate 300~400 ul of M 2 medium into the interspaces to form elliptical drops. Place a coverslip on top and gently push to make a seal. Fill around the drop with light mineral oil (Fig. 1 ).

4 . Fill the embryo fixation needle with fluorine and insert the needle into the tool sleeve, which is attached to the microinjector. Place the tool sleeve into the micromanipulator.

5 . Adjust the angle of the fixation needle so that the tip is parallel to the surface of the microinjection chamber.

6.Cover the blank area of the microscope stand with a piece of absorbent paper to prevent liquid from flowing out of the fixation needle and dripping into the microscope target.

7- Pull a sterilized Pasteur pipette to a length of 10 ~ 12c m and a diameter of about 0 -15m m to aspirate the D N A solution from the upper layer, avoiding particulate matter. Insert the dosing needle into the convex part of the microinjection needle and aspirate 2~5ul of D N A solution.

8. The DNA solution will not flow out of the syringe until all air bubbles have been removed. This process can be significantly accelerated by using the following method.

a- Attach a 20cc syringe with rubber tubing attached and a stopper on the rubber tubing and insert the thick end of the needle into the stopper.

b. Bend the rubber tubing so that the tip of the needle is pointing downward and apply the positive pressure created by gently tapping the tip of the needle to dislodge any air bubbles.

9. Insert the microinjection needle into the tool sleeve and place the tool sleeve into the micromanipulator. The tool sleeve should be lined up with the micromanipulator's retaining clips so that the microtool is centered just parallel to the long axis of the microinjection chamber. Adjust the tilt of the tool sleeve so that the microinjection needle is almost parallel to the surface of the microinjection chamber.

10 - Adjust the injection pressure (Pi) of the inflated microinjection needle to 70 ~ IOOhPa, and the fixation pressure (Ph) to approximately 50 % Pi.

11-Lift the tool sleeve in the microprocessor chamber until the tip of the needle is approximately 2 mm from the edge of the microinjection chamber, and then tighten the retaining clip.

12. Rinse the ova with a few drops of light mineral oil under M2 medium kept at 37°C in a CO2 incubator in CZB microtiter droplets.

1 3 . Remove the microinjection chamber from the microscope base and precipitate the ova on a plumb line in the center of the M2 droplet, starting at the midpoint of the droplet and extending to the top.

1 4 . At low magnification, advance the fixation needle and microinjection needle forward until they are visible in a small view as are the embryos. Keep the fixation needle separate from the injection needle to avoid damaging the tip.

1 5 . Rotate to the IOX objective and adjust the height of the fixation and microinjection needles until they are in the same focal plane as the embryo, repeating the adjustment until maximum magnification is achieved.

1 6 . The tip of the fixation needle should almost touch the surface of the injection chamber and should be in the same focal plane as the embryo. Adjust the angle of the fixation needle with respect to the slope of the f-plane so that the fixation needle moves without significant vibration.

Prokaryotic microinjection

1 7 . An egg is aspirated with a fixed needle and examined under high magnification. Only two prokaryotic nuclei should be visible in a fertilized egg, with one or more nucleoli within the prokaryotic nucleus. A single prokaryotic nucleus suggests that the egg has not been fertilized, and an egg with more than two prokaryotic nuclei will not develop properly.

1 8 . Focus on the microinjection needle and look for a very small swirling droplet at the tip of the needle when the injection function of the pneumatic syringe is activated. If you do not see one, it may be necessary to gently push it into the human fixed needle to lightly open the tip. If a large vortex is generated at the tip of the needle when the injection function is activated, the needle will cause excessive egg lysis. If the DNA solution is deficient or excessive, change the needle before proceeding.

1 9 . Adjust the position of the egg so that both the larger profile and the tip of the fixation needle are in quasi-focus.
The target prokaryotic nucleus should be located near the center of the egg, where the egg's plasma membrane will be difficult to penetrate. Ideally, the prokaryotic nucleus will be positioned so that the microinjection needle will not touch any of the nucleoli as it pierces the plasma membrane.

20. Ensure that the embryo is held firmly in place by the fixation needle, then place the injection needle until the tip is directly underneath the target nucleus (6 o' clock), and adjust the height of the needle (2-axis) until the microinjection needle and the membrane of the nucleus are in exactly the same focal plane. The prokaryotic membrane looks like a good loop, and the nucleolus is usually in the parafocal plane. The tip of the microinjection needle may be difficult to see clearly, so adjust the height of the needle until the upper and lower edges are sharp and visible, and then merge into one at the tip of the needle (Fig. 2, see color plate).

21. Place the tip of the needle in the opposite direction of the prokaryotic nucleus, aiming at the region without the nucleolus, and advance the zona pellucida and plasma membrane in a single smooth motion into the prokaryotic nucleus. Once when the puncture needle has penetrated the zona pellucida and plasma membrane, the surface of the egg will contract and then

22. If the needle fails to penetrate the egg plasma membrane or the prokaryotic membrane, small "bubbles" appear at the tip of the needle. If this occurs, withdraw the needle tip from the egg, place the prokaryotic membrane and the needle tip in the focal plane, and repeat the microinjection procedure.

2 3 . For microinjection, rinse the injected eggs with 5 to 6 drops of CO2-equilibrated C Z B medium ("Wash2" dishes) and incubate for 60 min ("Injected" dishes) to assess survival (these dishes were prepared in Step 4a of Scheme 3).

The survival of eggs after microinjection is dependent on individual skill and the strain of mouse used. Typically, greater than 90% of eggs are viable during microinjection.

Embryo Transfer material

reagents

anesthetic

Eggs/embryos that have been microinjected (as in protocol 4)

Ideally, eggs surviving a brief inoculation microinjection procedure are transferred into 〇.5dpc (days postcoitus) pseudopregnant recipient female mice. If female recipient mice are not available, the eggs are cultured in vitro in CZB medium to the multicellular embryonic stage.

Female recipient mice

Use 6 to 9 week old crosses (e.g. F1 or F2 C57BL/6XCBACa/J) or farrowing strains.

Oviductally ligated male mice

Instrumentation

Glass pipette (120~150u m )

Tool for unraveling the reproductive tubes of sham-fertilized mice

Stereo zoom dissecting microscope with fiber optic illumination for surgical embryo transfer (e.g. Leica M Z 7.5)
Surgical needle (9mm)

Methods

Preparation of sham-conceived pregnant female mice

1. The night before the embryo transfer procedure is performed, female recipients are paired with male mice with ligated vas deferens.

2 . The female mice were examined for the presence of a mating plug in the vagina by 12 noon the following day.
Female mice found to have a mating plug were terminated 0 - 5 dpc, because mating occurred during the dark cycle.
Transferring embryos to sham-fertilized recipient mice

3 . Anesthetize a 0.5 d p c sham-conceived recipient female mouse and prepare it for sterile surgery by removing one side of the reproductive tube by central abdominal excision.

4 . Add the egg/embryo to a 120 to 150 um glass pipette, punch or carefully tear the ovarian bursa with minimal bleeding, and transfer the egg/embryo to the uterine bursa opening.

5. If necessary, cut open the other side of the abdominal cavity and reproductive tract and repeat the above procedure.

6- Sew up the incision with a 9 mm surgical needle.

As a rule of thumb, 20% to 40% of microinjected embryos give rise to live fetuses, so transferring about 30 embryos to female recipients will produce the right number.

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