Rat somatic cell nuclear transplantation

Summary

Cloning rats is more difficult compared to mammals with mature cloning systems such as mice, pigs and cattle. The immature in vitro culture system of rat embryos is one of the reasons for the low development rate of rat cloned embryos. Naturally fertilized rat embryos often suffer from 2-cell block in existing culture systems, resulting in a low blastocyst rate. The extreme susceptibility of rat stage MII oocytes to spontaneous activation is another reason for the low development rate of rat cloned embryos. The protease inhibitor MG132, as the most widely used substance to inhibit spontaneous activation of rat oocytes, can effectively improve the cloning efficiency in rats. The technique of somatic cell nuclear transfer includes the main steps of donor cell preparation, denucleation of mature oocytes, nuclear transfer, and embryo transfer. Rat oocytes are very susceptible to spontaneous activation and therefore need to be inhibited during nuclear transfer.

Operation method

Rat somatic cell nuclear transplantation

Materials and Instruments

Equipment:
(1) Microscope operating platform:
By including inverted microscope (Leica, DMIRB, Germany)
Hydraulic microscope arm (NARISHIGW, 07003, Japan)
Hydraulic and pneumatic injection controller (Eppendorf, Cell-Tram, Germany)
Pizeo (PRIME TECH PMM, Japan) and a horizontal shock-absorbing table consisting of
②Syringes
③Aseptic surgical instruments
④Centrifuge
⑤CO
2
Incubator
Reagents:
①Material: rat
②Rat Embryo Culture Solution MRIECM
③Embryo manipulation solution M2
④ Proteasome inhibitor MG132
⑤ Hyaluronidase (hyaluronidase)
⑥ Endostatin I (butyrolactone I)
⑦ Strontium chloride (SrCl2)
⑧ colchicine (demecolcine)
⑨ Paraffin
⑩ cell relaxin B (eytochalasin B. CB)

Move

The basic process of nuclear transplantation of rat embryonic stem cells can be divided into the following steps:

(i) Preparation of nuclear transplantation related solutions

The relevant drugs and solutions used for nuclear transplantation of rat embryonic stem cells include: rat embryo culture medium MRIECM, embryo manipulation solution M2, proteasome inhibitor MG132, hyaluronidase (hyaluronidase), endostatin I (butyrolactone I), strontium chloride (SrCl2), colchicine (demecolcine). Strontium chloride (SrCl2), colchicine (demecolcine) and so on.

(ii) Preparation of donor cells

1. Select the rat embryonic stem cells on the 2nd day after passaging, which are in the period of vigorous growth, as the donor cells for nuclear transplantation.

2. Replace the culture medium with fresh one containing 0.05 μg/ml of the microtubule formation inhibitor colchicine for 3 hours.

3. The rat embryonic stem cells in suspension were aspirated and digested for 3 minutes, and a single-cell suspension was made using rat embryonic stem cell culture medium.

4. 50% to 80% of the rat embryonic stem cells are in the middle stage of cell division (MII stage) and placed on ice.

(iii) Acquisition of rat oocytes

1. Supernumerary ovulation, 12～16 hours after hCG injection in vivo, execute the rats by vertebral fracture method, open the abdominal cavity, and find the ovarian tissue.

2. The pot-belly of the oviduct containing the egg mass was cut off and placed into the preheated M2 in vitro operating solution with 5 μmol/L MG132 added.

3. Quickly cut through the tubal abdomen to free the egg mass.

4. Quickly transfer the egg mass into hyaluronidase containing 5 μmol/L MG132 and 0.1 mg/ml for 2 to 3 minutes.

5. Repeated blowing in combination with a mouth pipette was used to remove the granulosa cells encapsulated in the outer layer of the cumulus-oocyte complexes (COCs).

6. Oocytes were retransferred to 5 μmol/LMG132 of M2 in vitro operating solution and washed 2 to 3 times;

7. Place into pre-warmed rat embryo culture solution mR1ECM-1C microdrop containing 5 μmol/LMG132 and set aside.

(D) Nuclear transplantation operation

1. Add M2 operation solution containing 5 μmol/LMG132 to a homemade operating dish "Chamber" and cover it with liquid paraffin.

2. Blow the oocyte with a fixation needle to locate the genetic material inside the oocyte and fix the oocyte with the fixation needle when it is at the "3 o'clock" position of the oocyte.

3. Make a "hole" in the zona pellucida with a flat-bladed needle under the action of the Piezo.

4. The nuclear material of rat embryonic stem cells at MII stage is aspirated and injected directly into the cytoplasm, then the needle is withdrawn from the cytoplasm of the oocyte and the genetic material of the oocyte is removed.

5. The manipulated embryos were transferred back into microdrops of mR1ECM-1C culture medium and cultured for 30 minutes.

(E) Activation and culture of reconstituted embryos

1. mR1ECM-1C containing 150 μmol/L butyrolactone1 was used as the activation solution for rat reconstituted embryos for 2 hours.

2. Strontium chloride was used for solitary activation of oocytes. mRIECM-1C containing 10 mmol/L strontium chloride was used as the activation solution, and the activation time was 30 minutes. The addition of 5 mg/ml cytochalasin B (eytochalasin B. CB) was required for 6 hours to inhibit the expulsion of the dipolar body and the formation of diploid solitary embryos.

3. After the reconstructed embryos and orphan embryos were activated, they were transferred into rat mR1ECM-1C culture medium for culture, and the culture medium was replaced with fresh culture medium twice at 24 hours and 72 hours thereafter.

4. The development rates of 2-cell, 4-cell, mulberry, and blastocyst embryos were counted at 24, 72, 96, and 108 hours, respectively, after the completion of reconstruction.

(vi) Embryo transfer

1. Pseudo-pregnant rats were anesthetized with chloral hydrate at a concentration of 0.05 g/ml and injected intraperitoneally with chloral hydrate (1.5-2.0 ml per rat). 10 minutes later, the rats were fully anesthetized.

2. The backs of the mice were sterilized with 75% ethanol. After shaving, a cut was made on the dorsal side of the body to locate the oviduct/uterus, and the fat pads of the ovaries were gently clamped with clock forceps and the oviduct/uterus was pulled out.

3. The embryos were loaded into a mouth pipette under a body microscope. The mouth pipette was loaded according to the three-stage method: first, a small section of KSOM culture fluid was inhaled, then a small air bubble, then the embryos were aspirated, and after the embryos had been aspirated, another small air bubble and a small section of KSOM culture fluid were inhaled. The rat blastocysts to be transferred were injected into the uterine horns of 3.5-day sham pregnant rats.

4. After transplantation, the uterus/tubes are carefully placed back into the rat's abdominal cavity and surgically sutured. After implantation, the sham-pregnant rats were placed on a 37 ℃ hot plate to recover, and the rats woke up naturally after about 30 minutes.

5. After surgery, the rats were returned to the Laboratory Animal Center for further rearing.

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