Introduction

The technique described here is a slight modification (March 1997) of methods presented in:   Nagy A., J. Rossant. 1993. Production of completely ES cell-derived fetuses. In Gene targeting: a Practical Approach. (ed. A. Joyner). IRL Press at Oxford University Press.      To produce completely ES cell-derived embryos a clump of ES cells is sandwiched between two tetraploid embryos. For ES cell aggregation chimeras with diploid embryos, one embryo is aggregated with a clump of ES cells. Even in this case, the ES cell internalization is efficient enough to achieve a high level of ES cell contribution.   The benefit of single embryo aggregation is two fold:   First, double the number of aggregates can be produced in a single experiment. Second, we have found that the efficiency of germ line transmission of the targeted allele is better than with the sandwich type.   This observation may be explained as follows. When male (XY genotype) ES cells are aggregated with a female (XX genotype) morula, resulting in a phenotypically male, only primordial germ cells derived from the XY ES cells can contribute to the functional gametes. The XX primordial germ cells from the host embryo will not form gametes, therefore favoring the germ line contribution of ES cells containing the desired mutant allele.   This arrangement (XX host morula, XY ES cells) can occur in 50% of the single embryo aggregations, but only in 25% of the sandwich type aggregations. 

Flushing of eight-cell stage embryos  Materials: 

• • Dissecting microscope; 
• Flushing needle (The sharp tip of No. 30 G 1/2 needle is cut off and then rounded using sharpening stone); 
• 1 ml syringe; 
• Dissecting instruments (fine-pointed scissors, fine forceps); 
• No. 5 forceps (Dumont); 
• Mouth pipette (aspirator mouth piece, latex tubing, blue tip)  alternatively: 
• Finger controlled pipette (Manostat tubing, yellow tip, scalpel blade); 
• 9'' Pasteur pipettes; 
• 70% Ethanol; 
• Alcohol burner or Bunsen burner; 
• Sterile plastic Petri dishes (100 x 15 mm); 
• Sterile Organ Culture dishes (60 x 15 mm, Falcon, 3037); 
• M2 and KSOM media .

Method: 

1. The oviducts with the upper part of the uterus attached are removed from 2.5 days post-coitum (dpc) superovulated CD-1 females and placed into a drop of M2.    2. Under dissecting microscope the oviducts are flushed by inserting the flushing needle attached to a 1 ml syringe of M2 into the infundibulum.   3. The embryos are collected using mouth or finger controlled pipette and washed through several drops of M2 medium to remove any debris.   4.The embryos are washed in KSOM medium and cultured in organ culture dish in KSOM at 37o C, 5% CO2. 

Preparation of aggregation plate  Materials: 

• • Dissecting microscope; 
• Sterile tissue culture dishes (Easy Grip 35 x 10 mm, Falcon 3001-3); 
• KSOM medium; 
• light mineral oil (EMBRYO TESTED) (e.g. Sigma: M8410); 
• aggregation (darning) needle ( DN-09, BLS Ltd., Hungary ,) 
• 70% ethanol. 

Method: 

1. Place few rows of KSOM microdrops (roughly 3 mm in diameter) into tissue culture dish using syringe (e.g. 3 drops in the first and fourth and 4-5 in the second and third rows), cover with oil.   2. Sterilize aggregation needle by washing in ethanol.   3. Make six or more depressions in each microdrop (leaving a few drops intact for ES cell selection) by pressing the darning needle into the plastic and making slight circular movement. Do not twist the needle. This movement creates a tiny depression with clear smooth wall. It should be deep enough to hold the embryo.   4. Keep the plate at 37o C, 5% CO2. 

Removal of Zona Pellucida  Materials: 

• • Dissecting microscope; 
• Acid Tyrode's (Sigma: T1788); 
• Sterile plastic Petri dishes (100 x 15 mm); 
• M2 and KSOM medium in 1 ml syringes; 
• 9'' Pasteur pipettes; 
• Mouth or Finger controlled pipette. 

Method: 

1. Place a few drops of M2, KSOM and acid Tyrode's in the Petri dish.   2. Wash the group of embryos with as little medium as possible through one drop of Acid Tyrode's, then transfer to a fresh drop of the same solution.   3. Observe zona dissolution.   4. Immediately transfer the embryos into a drop of M2 medium as soon as the dissolution is completed.   5. Wash the embryos at least twice in KSOM drops before putting them in the aggregation plate.   6. Transfer embryos into aggregation plate by placing them one by one beside each depression, alternatively place one embryo inside each depression.   7. Prepare ES-cells for aggregation    by that time.  

ES cells/embryo aggregation  Materials: 

Dissecting microscope;   Prepared aggregation plate with depressions and embryos with removed zona;   Trypsinized ES-cells;   Mouth or Finger controlled pipette;   9'' Pasteur pipette.  

Method: 

1. Choose clumps of loosely connected ES cells and transfer them into microdrops (not containing embryos) of aggregation plate for final selection.   2. Select few clumps of ES cells (8 -15 cells in each); place each clump in a depression in the microdrop containing embryos.   3. Pick up the corresponding embryo and place it on the clump, alternatively, if the embryo is already inside the depression, place the clump of cells next to it.   4. Assemble all aggregates in this manner, check the plate, and culture overnight at 37o C, 5% CO2 .    The following morning, the majority of aggregates should have formed blastocysts. We transfer a maximum of 8-10 embryos into each uterine horn of a 2.5 dpc pseudopregnant recipient. Mature CD-1 females are used as pseudopregnant foster mothers and ordered at a weight of 30+ g.      In the event of a recipient shortage, it is possible:  

• to transfer up to 24-26 embryos per recipient; 
• to culture the aggregates (preferably morulae) for one more day and transfer them into 2.5 day pseudopregnant females; 
• to use 3.5 day pseudopregnant females. 

Transfer of embryos  Materials: 

• • 2.5 dpc pseudopregnant females; 
• Instruments: 
• Scissors; 
• Semken forceps (straight or curved with serrated tips); 
• Forceps with 1 x 2 teeth; 
• Dumont ss/mc or No. 5 forceps; 
• Serrefine (e.g.  Fine Scientific Tools: 18050-28 or 8051-28); 
• 26G1/2  or 30G1/2 needle; 
• Autoclip applier (Clay Adams B-D 763007); 
• Autoclips (Clay Adams B-D 7631); 
• Mouth controlled pipette; 
• M2 medium; 
• 2.5% Avertin: 
  • 2,2,2,-Tribromethanol  2.5 g (Morre-Tec Industries #1693); 
  • Tert-amyl alcohol  5.0 ml (Fisher: A-730-1). 

        Dissolve tribromethanol in Tert-amyl alcohol, then add to 200 ml distilled water. Place on a magnetic stirrer until solution is in one phase. Store in brown bottle and keep refrigerated until use. Should be warmed and shaken before use. Dosage is 0.2 ml/10 g body weight . 

Method: 

The embryo transfer procedure is described in details in many publications, such as the following:   1. Hogan, B., F. Constantini, E. Lacy. 1986. Manipulating the Mouse Embryo. Cold Spring Harbor, New York.      2. Bradley, A. 1987. Production and analysis of chimeric mice. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (ed. E.J.Robertson) IRL Press, Oxford, Washington, D.C.      3. Pappaioannou, V., R. Johnson. 1993. Production of chimeras and genetically defined offspring from targeted ES cells. In Gene Targeting: A Practical Approach (ed. A.Joyner) IRL Press at Oxford University Press.      4. Stewart C.L. 1993. Production of chimeras between embryonic stem cells and embryos. In Methods in Enzymology. vol.225. Academic Press Inc.