KR20010005424A - Method of cell fusion for producing a clone animal using somatic cell - Google Patents
Method of cell fusion for producing a clone animal using somatic cell Download PDFInfo
- Publication number
- KR20010005424A KR20010005424A KR1019990031528A KR19990031528A KR20010005424A KR 20010005424 A KR20010005424 A KR 20010005424A KR 1019990031528 A KR1019990031528 A KR 1019990031528A KR 19990031528 A KR19990031528 A KR 19990031528A KR 20010005424 A KR20010005424 A KR 20010005424A
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- South Korea
- Prior art keywords
- cell
- cells
- egg
- pipette
- solution
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Abstract
Description
본 발명은 복제동물을 생산하는 방법에 있어서의 세포융합방법에 관한 것으로서, 보다 상세하게는 체외수정(In Vitro Fertilization) 및 핵이식(Nuclear Transplantation) 기술에 의해서 예컨대 복제 소를 생산하는 방법에 있어서 체세포와 탈핵난자를 효과적으로 융합시키는 방법에 관한 것이다.The present invention relates to a cell fusion method in a method for producing a cloned animal, and more particularly, somatic cells in a method for producing a cloned cow by, for example, In Vitro Fertilization and Nuclear Transplantation techniques. And how to effectively fuse denuclearized eggs.
근래에 생명공학 또는 유전자 조작기술의 발달에 따라 유용한 작물들의 원하는 형질들을 조합한 다양한 재조합 생명체들의 다양한 생산 성공사례가 발표되어 왔고, 최근에는 예컨대 양 등과 같은 복제 동물들의 생산에 성공한 사례들이 속속 보고되었다. 복제동물의 생산은 현실적으로 생명공학 분야에서도 실로 고도로 축적된 기술적 뒷바침이 전제된 후에야 비로소 가능한 것이므로, 이러한 점에서 상기 복제동물의 생산은 당해 기술 수준의 척도가 되는 것이다.Recently, according to the development of biotechnology or genetic engineering technology, various production success cases of various recombinant organisms combining desired traits of useful crops have been published, and recently, success cases of producing cloned animals such as sheep have been reported one after another. . Since the production of cloned animals is practically possible only after a highly accumulated technical backing is premised in the field of biotechnology, the production of cloned animals is a measure of the technical level in this respect.
또한, 복제동물을 생산할 수 있는 능력 자체에는 의약학, 축산학 분야에서 장차 무한한 응용가능성이 잠재된 것으로 기대되고 있으므로, 복제동물의 생산은 기술혁신의 실로 획기적인 개가라고 할 수 있다. 이러한 복제된 소의 생산 방법의 근본 원리는 또한 다른 동물들을 복제하는데에도 응용될 수 있으므로, 향후 구체적 세부기술의 개발 여하에 따라 적용될 수 있는 동물의 범위도 넓어질 수 있을 것으로 전망된다.In addition, the ability to produce cloned animals is expected to have potential for future applications in the fields of medicine and animal husbandry. Therefore, the production of cloned animals is a breakthrough in innovation. Since the fundamental principle of the production method of the cloned cows can also be applied to clone other animals, it is expected that the range of animals that can be applied in the future according to the development of specific details.
이러한 점에 있어서, 본 발명의 목적은 체외수정 및 핵이식 기술에 의해서 복제된 동물, 특히 복제된 소, 및 이 복제된 소를 생산하는데 있어서, 공여 세포의 이식후에 세포를 융합시키는 효율적인 방법을 제공하는 것이다.In this regard, it is an object of the present invention to provide an efficient method of fusing cells after transplantation of donor cells in producing cloned animals, in particular cloned cattle, and the cloned cattle by in vitro fertilization and nuclear transfer techniques. It is.
본 발명자들은 상기 목적을 달성하기 위해 복제 소의 생산방법에 있어서 공여세포를 이식한 후, DC 조건의 전압의 경우 0.75∼2.25kv/cm으로 1∼2회에 걸쳐 회수×시간이 총 60㎲ 이내로 되도록 조정하여 전기 융합을 실시함으로써 성공적인 융합이 달성되는 것을 알게되어 본 발명을 완성하게 되었다.In order to achieve the above object, the present inventors transplanted donor cells in a method of producing a cloned cow, so that the number of times of recovery x time was within 60 ms in one or two times at 0.75 to 2.25 kv / cm in the case of DC condition voltage. It has been found that successful fusion is achieved by adjusting and conducting electrofusion to complete the present invention.
본 발명에 따르면, 다음과 같은 공정단계들로 구성되는 복제 소의 생산방법이 제공된다:According to the present invention, there is provided a method for producing a clone which consists of the following process steps:
(a) 도축난소로부터 미성숙난자를 채취 배양하는 단계;(a) harvesting immature eggs from slaughtered ovaries;
(b) 소 신체의 다양한 조직으로부터 세포주를 분리하여 보존하는 단계;(b) isolating and preserving cell lines from various tissues of the bovine body;
(c) 상기 (a)단계에서 준비된 성숙난자의 투명대의 일부를 절개하고 세포질의 10∼30%를 제거하여 탈핵을 실시하는 단계;(c) dissecting a portion of the zona pellucida of the mature egg prepared in step (a) and denuclearizing by removing 10-30% of the cytoplasm;
(d) 상기 (b)단계에서 준비된 공여세포를 상기 (c)단계의 핵이 탈핵된 세포에 이식하는 단계;(d) transplanting the donor cells prepared in step (b) into cells in which the nucleus of step (c) is denuclearized;
(e) 상기 (d)단계에서 이식 및 밀착시킨 작성란을 DC 조건의 전압으로 전기융합시키는 단계;(e) electrically fusion of the embryonated eggs implanted and adhered in step (d) to a voltage under DC conditions;
(f) 상기 융합된 세포를 융합과 동시에 또는 화학물질이 첨가된 배지내에서 활성화시키는 단계; 및(f) activating the fused cells concurrently with fusion or in a medium to which chemicals have been added; And
(g) 상기 활성화된 세포를 후활성화 및 미소적 배지내에서 배양시키는 단계이다.(g) culturing the activated cells in post-activation and micro medium.
본 발명의 바람직한 실시예에 따르면, 상기 (b) 단계의 세포는 소의 자궁관류액, 자궁내막, 난관에서 분리된 세포주이거나 난구세포일 수 있다.According to a preferred embodiment of the present invention, the cell of step (b) may be a cell line isolated from bovine uterine perfusate, endometrium, fallopian tube or cumulus cells.
본 발명의 다른 실시예에 따르면, 상기 (b) 단계의 세포는 소의 귀 또는 근육으로부터 분리된 세포이거나 태아섬유아세포이다.According to another embodiment of the present invention, the cell of step (b) is a cell isolated from a bovine ear or muscle or fetal fibroblasts.
또한, 상기 (b) 단계의 보존처리는 계대배양, 혈청기아배양 또는 동결보존에 의해 이루어지게 된다.In addition, the preservation of the step (b) is made by subculture, serum starvation or cryopreservation.
본 발명에 따르면, 상기 e) 단계에서 DC조건의 전압의 경우 0.75∼2.25Kv/cm로 1∼2회에 걸쳐 회수×시간 총 60us 이내로 조정하여 전기융합을 실시하는 세포융합법이 제공된다.According to the present invention, in the step e), the cell fusion method is performed in the case of the voltage under DC condition, which is adjusted to within 0.7us ~ 2.25Kv / cm within 1-2 times of recovery x time for a total of 60us.
본 발명의 다른 실시예에 따르면, 상기 방법중 어느 하나의 항의 방법에 따라 생산된 복제 소가 제공된다.According to another embodiment of the present invention there is provided a cloned cow produced according to the method of any one of the above methods.
이하에서는 본 발명 방법에 따라 수행된 바람직한 실시예 및 실험예를 구체적으로 기재한다.Hereinafter, the preferred examples and experimental examples performed according to the method of the present invention will be described in detail.
실시예 1 : 소의 체외수정(in vitro ferbilzation)Example 1 In Vitro Ferbilzation of Bovine
제1단계 : 체외성숙Stage 1: in vitro maturation
체외성숙(In vitro maturation)을 위한 배지(Medium)는 다음과 같이 준비하였다.Medium for in vitro maturation was prepared as follows.
배양(culture)용 TCM199(No. 3); 세척(washing)용-TCM199(No. 2);Culture TCM199 (No. 3); For washing-TCM199 (No. 2);
혈청(FBS, 1ml씩 분주되어 있는 것); E2(No. 7); FSH(No. 7)Serum (FBS, dispensed in 1 ml portions); E 2 (No. 7); FSH (No. 7)
상기 배양액을 준비하여 4-웰(well) 디쉬(dish)에 0.5㎕ E2을 분주하고, 알코올을 휘발시킨다. 12.5㎕ FSH(NO.7)을 분주하고 FBS 1㎖를 배양용 TCM199 9㎖에 첨가한 후 각각의 웰에 0.45mL씩 분주한다. 스퀴즈 보틀(Bottle)을 이용하여 웰 사이에 증류수를 분주한다. 35mm 디쉬에 남은 TCM 199 2.5ml를 분주하여 최종 세척액으로 사용한다.Prepare the culture solution and dispense 0.5 μl E 2 into a 4-well dish and volatilize the alcohol. Dispense 12.5 μl FSH (NO.7), add 1 mL of FBS to 9 mL of TCM199 for culture, and dispense 0.45 mL into each well. Distilled water is dispensed between the wells using a squeeze bottle. Dispense 2.5 ml of TCM 199 remaining in a 35 mm dish to use as a final wash.
난소채취는 생리식염수(35℃)와 가위, RP장갑, 장화등을 준비하여 도축장에서 난소를 채취한다.Ovarian collection is prepared by physiological saline (35 ℃), scissors, RP gloves, boots, etc. to collect the ovaries from the slaughterhouse.
검란준비를 위하여는 1cm 간격의 격자 눈금을 그은 100mm 디쉬와 세척용 TCM199을 2ml씩을 분주한 35mm 디쉬를 준비한다. 채취한 난소로부터 18G 니들(needle)이 장착된 10ml 주사기를 사용하여 2∼6mm의 난포를 흡입한다.To prepare the eggs, prepare a 100mm dish with grid scales of 1cm intervals and a 35mm dish dispensed with 2ml of TCM199 for washing. A 2-6 mm follicle is aspirated from the collected ovary using a 10 ml syringe equipped with an 18G needle.
다음에, 검란 및 세정을 수행한다. 먼저 난구세포가 충분히 부착되어 있고 세포질이 균질한 난자를 선발하고, 선발한 난자는 3회의 세정을 거쳐 최종 배양용 TCM199에 골라 놓고 각 웰 당 20∼50개의 난자를 35㎕씩 뽑아 분주한다.Next, the egg and washing are performed. First, eggs that are sufficiently adherent and have a homogeneous cytoplasm are selected, and the selected eggs are washed three times, selected in the final culture TCM199, and 35 μl of 20-50 eggs per each well are dispensed.
체외성숙을 수행함에 있어서는 5% CO2배양기내에서 24시간 배양한다.In vitro maturation is carried out in a 5% CO 2 incubator for 24 hours.
제2단계 : 체외수정Step 2: In Vitro Fertilization
체외수정(In vitro fertilization)은 다음과 같이 배지와 과정을 거쳐 수행하였다.In vitro fertilization was performed through the medium and process as follows.
배지 준비물은 Cap-TALP(뚜껑 열고 배양기, No. 1); IVF-TALP(뚜껑 닫고 배양기, No. 1); Washing-TALP(항온수조, No. 1); 헤파린(배양기, No. 8)로 하였다.Media preparations were Cap-TALP (lid open incubator, No. 1); IVF-TALP (cover closed and incubator, No. 1); Washing-TALP (hot water bath, No. 1); Heparin (cultivator, No. 8) was used.
수정과정(Procedure)은The procedure is
첫째, 미소적 작성 : 60mm 디쉬위에 43㎕ IVF-TALP 미소적(drop)을 만들고 인큐베이터(incubator)에 넣어둔다. 모든 미소적은 미네랄오일로 커버, 1개 미소적 당 5∼7개의 난자를 첨가함을 고려하여 적절한 수의 디쉬를 준비한다.First, micropreparation: Make a 43 μl IVF-TALP drop on a 60 mm dish and place in an incubator. Prepare the appropriate number of dishes, taking into account all the mineral mineral oil, adding 5-7 eggs per microdrop.
둘째, 스윔-업(Swim-up) : 5ml 둥근바닥 시험관(round bottom tube)를 10개 준비하여 cap-TALP를 0.8∼1ml씩 분주하고, 헤파린(Heparin)과 함께 배양기에 넣어둔다. 성숙 22시간에 정액 스트로우(straw)를 2∼3개 녹이고 파스퇴르 피펫을 사용하여 튜브 바닥에 약 0.1ml씩 분주, 배양기에 1시간동안 넣어둔다.Second, Swim-up: Prepare 10 5ml round bottom tubes and dispense 0.8 ~ 1ml of cap-TALP, and put them in the incubator together with Heparin. Two to three semen straws are dissolved at 22 hours of maturation, and a 0.1 ml aliquot is dispensed at the bottom of the tube using a Pasteur pipette and placed in the incubator for 1 hour.
셋째, 난자의 세정 : 스윔-업(Swim-up)을 하는 동안 3개의 35mm 디쉬에 세정용-TALP를 2ml씩 분주하여, 체외성숙중인 난자를 3회 세정한다. 세정한 난자는 작성한 각각의 미소적에 3㎕, 5∼7개씩 첨가한다.Third, egg washing: During washing-up, 3 ml of washing-TALP is dispensed into three 35 mm dishes, and the eggs are washed three times in vitro. 3 microliters and 5 to 7 eggs are added to each of the prepared microdrops.
넷째, 정자의 세정 : 스윔-업(Swim-up) 1시간후 정자 부유 상층액을 0.7∼0.8ml씩 흡입하여 15ml 튜브에 모은다.Fourth, cleaning of sperm: After 1 hour of swim-up (swim-up) inhaled sperm suspended supernatant by 0.7 ~ 0.8ml each collected in a 15ml tube.
모은 정자 부유액은 500∼700g, 6∼10분간 원심분리하고 100∼200㎕의 정자 펠릿(pellet)을 남긴 후 상층액을 제거하고 Cap-TALP로 1∼2회 더 원심세정한다.The collected sperm suspension is centrifuged for 500 to 700 g for 6 to 10 minutes, leaving 100 to 200 μl of sperm pellets, and then the supernatant is removed and centrifuged once or twice with Cap-TALP.
최종원심분리 후 상층액을 제거하고 약 100㎕의 정자 펠릿을 남긴다.After final centrifugation, the supernatant is removed and about 100 μl of sperm pellets are left.
다섯째, 정자농도의 결정: 4% HCl 190㎕와 정자 펠릿 10㎕를 섞은 후 혈구계산반의 양쪽에 적당량을 주입하여 100∼400배의 배율하에서 정자의 수를 센다. 5개의 큰 구획중 가운데의 구획에서(이 구획은 25개의 소구획으로 나뉘어져 있다) 5군데(네 모서리와 한가운데)에 있는 정자를 세고 나머지 한쪽 계산반의 것도 동일한 방법으로 센다. 이렇게 모두 10군데를 센 후, 총 100개가 넘으면 정자 펠릿의 부피(마이크로 피펫으로 100㎕전후로 조정하면서 측정한다.) × 총 개수 ÷ 100을 한 수가 첨가할 헤파린의 양이고, 이 수치에서 펠릿의 부피을 뺀 수가 추가로 첨가할 cap-TALP의 양이 된다(예: 카운팅한 개수가 130개이고 펠릿이 90㎕이면 90×130/100=117이 첨가할 헤파린의 양이고 117∼90이 추가로 첨가할 Cap-TALP의 양이다.)Fifth, the determination of sperm concentration: After mixing 190 μl of 4% HCl and 10 μl of sperm pellet, inject an appropriate amount into both sides of the hemocytometer and count the number of sperm under the magnification of 100 to 400 times. In the middle of the five large compartments (which are divided into 25 subdivisions), count the sperm in five places (in the middle and at the four corners) and count in the same way as the other one. After counting all 10 places, if the total number is more than 100, the volume of sperm pellets (measured by adjusting 100 μl before and after with a micropipette) × total number ÷ 100 is the amount of heparin to be added. The subtracted amount is the amount of cap-TALP to be added (for example, if the counted number is 130 and the pellet is 90 µl, 90 × 130/100 = 117 is the amount of heparin to be added, and caps 117 to 90 are to be added). The amount of -TALP.)
수정과정에서 수정능 획득을 위해 헤파린(최종농도 100㎍/ml)을 첨가하였으며, 15분간 배양기에서 배양하여 수정능 획득을 유도하였다.Heparin (final concentration 100µg / ml) was added to obtain fertility during fertilization, and fermentation was induced by incubation in incubator for 15 minutes.
또한, 정자를 4㎕씩 미소적내에 주입(최종 정자농도는 2×106개/ml)하고 5% CO2배양기내에서 18∼30시간 배양하였다.In addition, 4 μl of sperm was injected into the microspheres (the final sperm concentration was 2 × 10 6 cells / ml) and incubated for 18 to 30 hours in a 5% CO 2 incubator.
제3단계 : 체외배양Step 3: In Vitro Culture
체외 배양(In vitro cultivation)은 다음과 같이 준비하여 수행하였다.In vitro cultivation was performed as follows.
배지(Medium) 준비물은 mTALP(incubator, No. 11); 세정용 배양액 (washing-TCM 혹은 washing-TALP)(water bath)으로 하였다.Medium preparations were mTALP (incubator, No. 11); Washing culture (washing-TCM or washing-TALP) (water bath) was used.
그 과정(Procedure)은 먼저, 미소적을 작성한다. 35mm 혹은 60mm 디쉬, 25∼30㎕ mTALP 미소적, 인큐베이터(incubator)에 보관(1개 점적 당 5∼10개의 난자를 첨가함을 고려하여 적절한 수의 디쉬 준비)하고, 35mm 디쉬 3개에 세정용 배양액을 2ml씩 분주, 가온판(warm plate)위에 올려 놓는다.The procedure first creates a smiley. 35 mm or 60 mm dish, 25-30 μl mTALP micron, stored in an incubator (prepared the appropriate number of dishes considering the addition of 5 to 10 eggs per drip), for washing on three 35 mm dishes Dispense 2 ml of the culture and put it on the warm plate.
다음에, 난구세포제거(Denuding)과정으로 세정용 배양액이 들어있는 15ml 시험관(tube)에 체외수정된 난자를 넣어 2분간 와동(vortexing)하거나 mouth pipetting하여 난구세포를 제거한다.Next, in vitro fertilized eggs are placed in a 15 ml tube containing a washing medium for denuding, followed by vortexing or mouth pipetting for 2 minutes to remove the cells.
난구세포가 제거된 난자를 3회 세정, 5㎕에 5∼10개씩 흡입하여 미소적에 주입한 후, 5% CO2배양기 또는 5% CO2, 5% O2, 90% N2에서 5∼8일간 배양하되, 체외수정 시작시기부터 계산하여 48시간 전후로 검란, 발육단계별로 구분하여 미소적내에 배치한다.Wash the eggs from which the oocytes had been removed three times, inject 5-10 pieces into 5 µl, and inject them in small amounts. Then, 5-8 in a 5% CO 2 incubator or 5% CO 2 , 5% O 2 , 90% N 2 . Incubate daily, and calculate from the beginning of in vitro fertilization and place them within the microspheres by dividing the egg and the stage of development around 48 hours.
최종 검란은 수정 7일에 실시하여 후기배 발육율 계산한다.Final eggs should be fertilized at 7 days of fertilization to calculate late embryonic development.
제4단계 : 수정란 동결(Embryo Freezing, 기기-FHK, ET1N)Stage 4: Embryo Freezing (Embryo Freezing, Device-FHK, ET1N)
수정란 동결을 위한 동결배지(Freezing Medium)로 PBS(+)(표.9) 2.25ml와 혈청(FBS) 2.25ml를 35mm 디쉬에 함께 분주하여 희석한 후 글리세롤(Sigma, G-9012)을 0.5ml 첨가(10% 글리세롤)한다.2.25ml PBS (+) (Table 9) and 2.25ml serum (FBS) are added together in a 35mm dish and diluted 0.5ml of glycerol (Sigma, G-9012). Add (10% glycerol).
다음에는 동결기를 가동하여 -5℃를 유지시키고, 동결할 수정란을 선발하여 PBS(+)로 세정하고 동결배지에 넣어 15∼20분간 정치시킨다.Next, the freezer is operated to maintain -5 ° C, the embryos to be frozen are selected, washed with PBS (+), placed in a freezing medium and allowed to stand for 15 to 20 minutes.
그후, 0.25ml 스트로우에 수정란을 장착(양 끝단에 공기층을 2층씩 만들고 가운데에 수정란이 포함된 배양액을 흡입, 봉인은 불에 달군 집게(forcep)를 사용하여 열밀봉(heat sealing)한다.Then, fertilized eggs are placed on a 0.25 ml straw (two layers of air at both ends, aspirated from the culture solution containing fertilized eggs in the middle, and sealed by heat sealing using forceps.
즉, 면봉-(배양액)-공기층-(배양액)-공기층-(배양액+수정란)-공기층-(배양액)-공기층-(배양액)-열 밀봉(heat sealing)으로 수행한다.That is, cotton swab-(culture liquid)-air layer-(culture medium)-air layer-(culture medium + fertilized egg)-air layer-(culture medium)-air layer-(culture medium)-heat sealing.
그리고, -5℃에서 동결기에 스트로우를 장착(면봉이 아래쪽)하고, 5분간 이 온도를 유지시킨 다음, 액체 질소탱크에 집게(forcep)를 10∼20초간 넣었다 빼어서 스트로우의 상부 2번째 공기층(밑줄그은 부분)의 하단을 살짝 집어주어 식빙(seeding)시킨다.Then, the straw is attached to the freezer at -5 ° C (a cotton swab is lowered), and maintained at this temperature for 5 minutes, and then a forcep is put into a liquid nitrogen tank for 10 to 20 seconds, and the upper second air layer of the straw ( Gently pinch the bottom of the underlined part to seeding.
식빙 직후 0.3℃/min의 비율로 -30℃까지 온도를 하강시킨 다음 -30℃가 되면 10분간 온도를 유지시킨 후 액체 질소탱크내에 동결보존한다.Immediately after planting, the temperature is lowered to -30 ° C at a rate of 0.3 ° C / min. When the temperature reaches -30 ° C, the temperature is maintained for 10 minutes and then cryopreserved in a liquid nitrogen tank.
제5단계 : 동결란 융해(Thawing)Step 5: Thawing Frozen
동결란을 융해하기 위해 다음과 같이 배지를 준비하였다. 6% 글리세롤, 3% 글리세롤 및 0% 글리세롤 용액; 수정란이식 배지(Embryo transfer medium)- PBS(+), FBS(1ml씩 분주된 것).The medium was prepared as follows to thaw the frozen eggs. 6% glycerol, 3% glycerol and 0% glycerol solution; Embryo transfer medium-PBS (+), FBS (1 ml aliquots).
다음, 동결란을 융해하기 위하여는, PBS(+) 4ml에 FBS를 1ml 첨가하여 20% FBS 용액을 제조한 다음, 융해 용액 및 20% FBS 첨가한 PBS(+)용액을 35mm 디쉬에 각각 분주하고 라벨링(labeling)한다.Next, in order to thaw the frozen eggs, 1 ml of FBS was added to 4 ml of PBS (+) to prepare a 20% FBS solution. Then, the fusion solution and the 20% FBS-added PBS (+) solution were respectively dispensed and labeled in a 35 mm dish. (labeling)
그리고, 약 30℃의 온수를 준비(직경이 20cm이상인 용기사용)하여, 액체 질소탱크로부터 동결 스트로우를 꺼내 공기 중에서 약 5초간 정치한 후, 온수에 약 30초간 담가서 융해(흔들거나 세우지 말 것)시킨다.Then, prepare a hot water of about 30 ℃ (using a container with a diameter of 20cm or more), take out the freeze straw from the liquid nitrogen tank, leave it in air for about 5 seconds, soak it in hot water for about 30 seconds to melt (do not shake or stand). Let's do it.
그 후, 6% 글리세롤 용액에 스트로우내의 수정란을 침적시키고 5분간 정치시킨 다음, 3% 글리세롤용액에 스트로우내의 수정란을 침적시키고 5분간 정치시킨 후, 0% 글리세롤용액에 스트로우내의 수정란을 침적시키고 5분간 정치시킨 다음, 수정란 이식배지(Embryo transfer medium)에 수정란을 침적시킨 후 스트로우에 장착하고, 수정란이식을 실시한다.Subsequently, the fertilized egg in the straw was immersed in a 6% glycerol solution and allowed to stand for 5 minutes. Then, the fertilized egg in the straw was immersed in a 3% glycerol solution and allowed to stand for 5 minutes. After standing still, the fertilized egg is deposited on the embryo transfer medium (embryo transfer medium), mounted on the straw, and fertilized egg transplant.
제6단계 : 난자 염색(Aceto-orcein 이용)Step 6: Oocyte Staining (using Aceto-orcein)
난자염색을 위한 준비물(Preparation)로서 고정액 (Carcoddylate buffer,조직절편고정용액)을 사용하고, DW, 에탄올, Aceto-orcein 용액(아세트산 45ml + orcein 1g + 증류수 55ml), 슬라이드 글라스(슬라이드 글라스), 커버 글라스(커버글라스), 바세린 파라핀 용액을 준비하였다.Using the fixed solution (Carcoddylate buffer, tissue section fixation solution) as preparation for egg staining, DW, ethanol, Aceto-orcein solution (45 ml of acetic acid + 1 g of distilled water + 55 ml of distilled water), slide glass (slide glass), cover A glass (cover glass) and petrolatum paraffin solution were prepared.
난자염색과정(Procedure)은 다음과 같다.The egg staining process (Procedure) is as follows.
즉, 슬라이드글라스의 1/3 부위에 4군데(커버글라스의 모서리에 닿을 정도) 파라핀 점적을 형성시킨 다음, 난자를 4군데의 점 가운데 마우스피펫으로 4∼10개 정도 올려놓고 커버글라스를 덮는다.That is, four (approximately the edge of the cover glass) paraffin droplets are formed on one-third of the slide glass, and then 4 to 10 eggs are placed in the four spots with a mouse pipette to cover the cover glass.
다음, 볼펜이나 피펫팁 등의 가는 물건을 이용하여 난자를 누른다.Next, press the egg using a thin object such as a ballpoint pen or a pipette tip.
그리고, 고정액을 흘려 보내고 거름종이를 반대편에 대어 고정액 통과 후 10∼15분간 정치(습윤유지)시킨 다음, DW를 통과시킨다.Then, the fixed liquid is flowed out, the filter paper is placed on the other side, and then passed through the fixed liquid for 10 to 15 minutes (wet maintenance), and then passed through the DW.
그 후, 에탄올을 통과시킨 다음, Aceto-orcein 으로 통과하여 염색시킨다.Thereafter, ethanol was passed through, followed by dyeing with aceto-orcein.
그리고, 현미경하에서 관찰한다.And it observes under a microscope.
제7단계 : 난자 및 수정란의 염색(Giemsa Stain 이용)Step 7: dyeing eggs and fertilized eggs (using Giemsa Stain)
난자와 수정란의 염색을 위하여 다음과 같이 준비하였다. 저장처리용액 (0.9% 시트르산 나트륨 용액 또는 0.56% KCL 용액); 고정액Ⅰ(메탄올:빙초산:증류수 = 5 : 1 : 4); 고정액Ⅱ( 메탄올:빙초산 = 3 : 1); 5% Giemsa; 슬라이드 글라스.For staining of eggs and fertilized eggs were prepared as follows. Stock solution (0.9% sodium citrate solution or 0.56% KCL solution); Fixed solution I (methanol: glacial acetic acid: distilled water = 5: 1: 4); Fixed solution II (methanol: glacial acetic acid = 3: 1); 5% Giemsa; Slide glass.
염색과정(Procedure)은 다음과 같다.The procedure is as follows.
수정란을 저장용액에 15분간 침지시켜 수화시킨 다음, 가능한 적은 용량의 저장액과 함께 수정란을 슬라이드 글라스 위에 점적시키고, 수정란위에 마이크로피펫(micro 피펫)을 사용하여 고정액Ⅰ의 점적(zona pellucida를 제거)을 형성시킨다.Immerse the fertilized egg in the stock solution for 15 minutes to hydrate it, and then place the fertilized egg on the slide glass with the smallest amount of stock solution, and use a micro pipette on the fertilized egg to remove the drop of fixative I (zona pellucida). To form.
고정액Ⅱ를 점적하여 슬라이드 글라스에 고정시키고 공기중에서 건조시킨다.Dropping solution II is fixed on the slide glass and dried in air.
Giemsa 용액내에서 15분간 염색하고,Stain for 15 minutes in Giemsa solution,
증류수를 이용하여 여분의 염색액을 슬라이드에서 제거한다.Remove excess dye from the slide with distilled water.
다음, 저배율(4×10)로부터 고배율(10×40)로 관찰한다.Next, observation is performed from low magnification (4 × 10) to high magnification (10 × 40).
제8단계 : 체세포의 핵상관찰(Giemsa Stain 이용)Step 8: nuclear observation of somatic cells (using Giemsa Stain)
Giemsa stain을 이용하여 체세포의 핵상관찰을 위해 다음 준비물을 사용하였다. 즉, 저장처리용액 (0.9% sodium citrate 용액 또는 0.56% KCL 용액); 고정액 (메탄올 : acetic acid = 3 : 1); 5% Giemsa; 슬라이드 글라스; 15ml 시험관The following preparations were used for nuclear observation of somatic cells using Giemsa stain. That is, a stock solution (0.9% sodium citrate solution or 0.56% KCL solution); Fixative (methanol: acetic acid = 3: 1); 5% Giemsa; Slide glass; 15ml test tube
체세포의 관찰과정으로는,In the process of observing somatic cells,
디쉬내의 세포를 부유시킨 후(Single cell), 15ml 시험관에서 1500rpm으로 2분간 원심분리하여 세포 펠릿(pellet)을 얻는다.After floating the cells in the dish (Single cell), centrifuged for 2 minutes at 1500rpm in a 15ml test tube to obtain a cell pellet (pellet).
다음, 펠릿(pellet)에 저장액을 첨가하고 부유하여 15분간 수화시킨 다음 총 부피의 1/3에 해당하는 고정액을 첨가한다.Next, the stock solution is added to the pellet, suspended, hydrated for 15 minutes, and then a fixed solution corresponding to 1/3 of the total volume is added.
그 후, 원심분리시킨(1500rpm, 2분) 다음, 고정액으로 세척하고(원심분리→ 상층액 제거→재부유, 3회), 마지막 원심분리 후 적정농도가 되도록 고정액을 남겨 세포 부유 슬라이드위에 점적(최대한 확산하도록)시키고나서, 공기중에서 건조시킨다.Then, centrifuged (1500 rpm, 2 minutes), and then washed with a fixed solution (centrifugation → supernatant removal → resuspension, 3 times), leaving a fixed solution to a titration concentration after the last centrifugation and dropping onto a cell suspension slide ( Spread as much as possible, then dry in air.
그리고 Giemsa 용액내에서 15분간 염색시킨 다음, 증류수를 이용하여 여분의 염색액을 슬라이드에서 제거한다.After dyeing for 15 minutes in Giemsa solution, the excess dye solution is removed from the slide using distilled water.
그 후, 저배율(4×10)에서 고배율(10×40)로 관찰한다.Then, it observes with high magnification (10x40) at low magnification (4x10).
실시예 2 : 소의 핵이식Example 2 Bovine Nuclear Transplantation
핵 이식준비 과정은 다음과 같다.The nuclear transplant preparation process is as follows.
제1단계 : Micro 피펫의 준비Step 1: Preparation of the Micro Pipette
Micro피펫을 준비함에 있어서 사용기기는,In preparing the micro pipette,
Puller(Narishige PC-10), Microforge(Narishige MF-9), Glinder(Narishige EG-4) 등이다.Puller (Narishige PC-10), Microforge (Narishige MF-9), Glinder (Narishige EG-4).
우선, 절개 및 탈핵용 피펫(cutting & Enucleation 피펫)제조하기 위하여, 유리관(Drummond; Sigma, P-2174 or narishige)을 사용하여 추(Light 1개와 heavy 1개 또는 light 2개, heavy 1개)를 부착하고 Pulling step으로 Step 2에 고정한 다음, 온도를(heat 1- 101, heat 2- 101.5) 조정한다.First, in order to manufacture a cutting and denuclearization pipette (cutting & Enucleation pipette), a weight (1 light, 1 heavy or 2 light, 1 heavy) is used using a glass tube (Drummond; Sigma, P-2174 or narishige). Attach and fix to Step 2 with the Pulling step, then adjust the temperature (heat 1-101, heat 2-101.5).
두번째로, 공여핵세포 이식용 피펫(Injection pipette, 내경 30∼40㎛)제조하기 위하여는 유리관(Drummond; Sigma, P-2174 or narishige)을 사용하여 추(Light 1개) 또는 추를 제거하고 Pulling step으로 Step 2에 고정한 다음 온도를(heat 1∼101, heat 2∼2 101.5) 조정한다.Second, in order to manufacture injection pipette (injection pipette, internal diameter 30-40㎛), remove weight (1 light) or weight by using glass tube (Drummond; Sigma, P-2174 or narishige) and pulling Fix to Step 2 as a step and adjust the temperature (heat 1 ~ 101, heat 2 ~ 2 101.5).
다음에, 고정용 피펫(Holding pipette, 내경 70∼130㎛)제조(2가지)하기 위하여는 다음과 같이 하였다.Next, in order to manufacture (holding pipettes (Holding pipette, inner diameter 70 ~ 130㎛) (two kinds)) as follows.
i) 유리관(Drummond; Sigma, P-2174)를 사용하는 경우 :i) When using glass tubes (Drummond; Sigma, P-2174):
추(Light 1개 ∼light 2개, heavy 1개)를 부착하고 Pulling step(Step 1)에 고정한 다음 온도를 heat 2∼101.5으로 조정한다.Attach the weight (1 light ~ 2 light, 1 heavy), fix it to the pulling step (Step 1), and adjust the temperature to heat 2 ~ 101.5.
ii) 유리관(Narishige G-1 or GD-1)을 사용하는 경우ii) When using glass tubes (Narishige G-1 or GD-1)
추(Light 1개, heavy 1개)를 부착하고 Pulling step(Step 1)에 고정한 다음, 온도는 heat 2∼101.5로 조정한다.Attach the weight (1 light, 1 heavy) and fix it to the pulling step (Step 1), then adjust the temperature to heat 2 ~ 101.5.
넷째로, 난자의 세정 및 운반용 피펫 제조하기 위하여Fourth, to prepare a pipette for cleaning and transporting eggs
알코올 램프를 이용하여 내경이 300㎛ 이상이 되도록 하고, 사용시 한번 이용한 것은 폐기한다.Use an alcohol lamp so that the inner diameter is 300 µm or more, and discard the used one at the time of use.
다섯째로, 난구세포제거(Denuding용)용 피펫을 제조하기 위하여는Fifthly, in order to manufacture a pipette for denuding
알코올램프를 이용하여 내경이 약 130㎛인 것을 여러개 만들어 준비한다.Using alcohol lamp, prepare several things with inner diameter of about 130㎛.
제2단계 : 배양액의 조제Second Step: Preparation of Culture Solution
배양액(medium)은 다음과 같이 준비하였다.Culture medium was prepared as follows.
IVM용 TCM-199으로 체외수정과 동일하게 제조하고, TCM-W으로, 사용 당일에는 작업용 미소적을 작성하되 9ml의 TCM-W에 1ml의 FBS(FCS)를 넣어 10%FBS TCM-W를 만든 후, 필요한 점적 수만큼 100㎕ 피펫을 이용하여 20∼30㎕ 점적을 적정한 크기(35mm, 60mm, 100mm등)의 디쉬에 만들고, 미네랄 오일로 도포한 다음 사용한다.TCM-199 for IVM is manufactured in the same way as in vitro fertilization, and on the day of use with TCM-W, a small amount for work is prepared, but 1ml of FBS (FCS) is added to 9ml of TCM-W to make 10% FBS TCM-W. Using a 100µl pipette, use 20µl droplets in a suitable size (35mm, 60mm, 100mm, etc.) dish, apply with mineral oil, and use.
또, 하이알루로니데이즈(Sigma, H-3506) solution을 제조하기 위해서는, 5ml의 TCM-W에 하이알루로니데이즈(Hyaluronidase) 0.0500g을 첨가하여 원액(stock)을 만든다. 만들어진 원액은 111㎕씩 eppendorf 시험관에 분주하여 냉동실에 보관한다. 사용시에는 TCM-W를 1ml 분주하여 최종농도를 0.1%로 맞추어 35mm 디쉬에 분주하여 난구세포제거(denuding)에 이용한다.In addition, in order to prepare a hyaluronidase (Sigma, H-3506) solution, 0.05ml of hyaluronidase is added to 5 ml of TCM-W to make a stock (stock). The prepared stock solution is dispensed into eppendorf test tubes by 111 µl and stored in the freezer. In use, 1 ml of TCM-W is dispensed and the final concentration is adjusted to 0.1% and dispensed into 35 mm dishes for dendritic cell removal.
그리고, Cytochalasin B(sigma, C-6762) solution 제조를 위해서는,And, for the preparation of Cytochalasin B (sigma, C-6762) solution,
DMSO(Sigma, D-5879) 133㎕에 1mg의 사이토칼라신 B(cytochalasin B)를 녹이면 농도는 7.5mg/ml이 되는데, 이것을 eppendorf 시험관에 1㎕씩 분주해서 냉동실에 보관한다. 사용할때는 여기에 10% FBS(또는 FCS)첨가 TCM-W를 1ml 첨가하여 절개 및 탈핵(cutting & enucleation) 미소적 제작에 이용한다.After dissolving 1 mg of cytocalasin B in 133 μl of DMSO (Sigma, D-5879), the concentration is 7.5 mg / ml. Dispense 1 μl into eppendorf test tubes and store in the freezer. In use, 1 ml of 10% FBS (or FCS) -added TCM-W is added and used for microfabrication of cutting and enucleation.
또한, PHA-P(Phytohematoglutinin: L-9132) solution 제조법은 5mg의 PHA를 10ml의 TCM-W와 섞은 다음 100㎕씩 eppendorf 시험관에 분주한 다음, 실험에 사용할 경우 eppendorf 시험관에 400㎕의 TCM-W를 첨가하여 작업용 미소적을 만들어 사용한다. 이때 최종 농도는 100㎍/ml이 되도록 한다.In addition, the PHA-P (Phytohematoglutinin: L-9132) solution was prepared by mixing 5 mg of PHA with 10 ml of TCM-W and dispensing 100 μl into eppendorf test tubes, and then 400 μl of TCM-W into eppendorf test tubes when used in the experiment. Use it to make working microdroplets by adding. At this time, the final concentration is to be 100 ㎍ / ml.
그리고, 전기융합 및 활성화 배지(Electrofusion and Activation media) 제조를 위해서는 Mole 농도에 따른 만니톨(Sigma, M-1902)의 첨가량 (100ml기준)을 0.28M 만니톨: 5.1016g; 0.25M 만니톨: 4.555g; 0.26M 만니톨: 4.7372g; 0.1mM MgSO4, 0.1mM CaCl2포함하고, 기타 첨가물(만니톨 mole 농도에 관계없음): 0.1mM MgSO4또는 MgCl2, 0.1mM CaCl2(융합과 활성 동시군에서 첨가); Hepes 0.5mM; BSA 0.05%을 첨가하되 DW를 100ml까지 첨가한다.And, in order to prepare the electrofusion and activation media (Electrofusion and Activation media) the amount of mannitol (Sigma, M-1902) added according to the Mole concentration (100ml standard) 0.28M mannitol: 5.1016g; 0.25 M mannitol: 4.555 g; 0.26 M mannitol: 4.7372 g; 0.1 mM MgSO 4 , 0.1 mM CaCl 2 , other additives (regardless of mannitol mole concentration): 0.1 mM MgSO 4 or MgCl 2 , 0.1 mM CaCl 2 (added in the fusion and active co-group); Hepes 0.5 mM; Add 0.05% BSA but add up to 100 ml DW.
pH를 7.2∼7.4로 맞추고, 5ml 시험관에 약 4ml씩 분주하여 냉동(장기간) 혹은 냉장실(단기)에 보관한다.Adjust the pH to 7.2-7.4, dispense about 4ml into 5ml test tube and store in freezer (long-term) or freezer (short-term).
한편, DMAP(Sigma, D-2629)제조법은 10ml IVC용액에 0.0031g DMAP을 첨가하여 용해 및 여과후(0.22㎛ filter 사용) eppendort 시험관에 분주시키고 냉동보관(1.9mM)한다.On the other hand, in the manufacturing method of DMAP (Sigma, D-2629), 0.0031 g DMAP is added to a 10 ml IVC solution, dissolved and filtered (using a 0.22 μm filter), and then dispensed into a eppendort test tube and frozen (1.9 mM).
또, Cycloheximide(Sigma, C-7698)는, Ethanol 100ml에 Cycloheximide 1g을 녹여 ×1000배 Stock을 작성 최종농도10㎍/㎖가 되도록 IVC medium에 첨가하여 사용한다.Cycloheximide (Sigma, C-7698) is prepared by dissolving 1 g of Cycloheximide in 100 ml of ethanol and adding a 1000 × stock to the IVC medium to a final concentration of 10 µg / ml.
그리고, Ionomycin(Sigma, I-0634)는, DMSO(Sigma, D-5879) 1.34ml에 1mg의 Ionomycin를 녹여 1mM 원액을 제조한 다음, 최종농도 5μM 이 되도록 1% BSA TCM-W 또는 IVC medium내에 첨가하여 사용하고 원액을 냉동보관한다.Ionomycin (Sigma, I-0634) was dissolved in 1mg of Ionomycin in 1.34ml of DMSO (Sigma, D-5879) to prepare 1mM stock solution, and then in 1% BSA TCM-W or IVC medium to a final concentration of 5μM. Add to use and freeze the stock solution.
Ca2+Ionophore(A23187)는, DMSO(Sigma, D-5879) 1.66ml에 1mg의 Ca2+Ionophore(A23187)를 녹여 1mM 원액 제조시 최종농도 5μM 가 되도록 1% BSA, TCM-W 또는 IVC medium내에 첨가하여 사용하고 원액을 냉동보관한다.Ca 2+ Ionophore (A23187) is dissolved in 1 mg of Ca 2+ Ionophore (A23187) in 1.66 ml of DMSO (Sigma, D-5879), so that 1% BSA, TCM-W or IVC medium is used to produce a final concentration of 5 μM. It is added and used in the container, and the stock solution is kept frozen.
배양액(culture Medium)은 mTALP (No. 11), mSOF (No. 12), CR2aa (No. 13), 난관공배양(No. 14)중에서 선택하고, Hoechst 33342 (Sigma, B-2261)는 25mg의 Hoechst에 증류수를 5ml 섞어 희석(5mg/ml 의 stock)시킨 다음, Stock 1∼2㎕씩 eppendorf 시험관에 분주, 냉동보관한다. 그리고, 사용시에는 시험관에 10% FBS 첨가하고 TCM-W를 1ml 첨가하여 희석시킨 후 사용(최종농도 5∼10㎍/ml)한다.Culture medium was selected from mTALP (No. 11), mSOF (No. 12), CR2aa (No. 13), fallopian tube culture (No. 14), Hoechst 33342 (Sigma, B-2261) 25mg Dilute (5mg / ml stock) with 5ml of distilled water in Hoechst of the mixture, and dispense 1 ~ 2μl of stock into eppendorf test tube and store frozen. In addition, 10% FBS is added to a test tube, 1 ml of TCM-W is diluted, and it is used after use, and it uses (final concentration 5-10microgram / ml).
10∼15분간 정치한 다음 암실, 형광현미경으로 탈핵여부를 판단할 수 있다.After standing for 10-15 minutes, denuclearization can be determined using a dark room or a fluorescence microscope.
Nocodazole stock (Sigma, M-1404)은, DMSO(Sigma, D-5879) 2.5ml를 10mg의 nocodazole병에 넣어 희석시킨 다음 50∼100㎕씩 eppendorf 시험관에 분주(4mg/ml의 stock)하고 Stock 1㎕를 원하는 배양액 10ml에 첨가(0.4㎍/m)한다.Nocodazole stock (Sigma, M-1404) was diluted in 2.5 ml of DMSO (Sigma, D-5879) in a 10 mg nocodazole bottle, and then divided into 50 to 100 μl in eppendorf test tubes (4 mg / ml stock). [Mu] l is added (0.4 [mu] g / m) to 10 ml of the desired culture.
Acid Tyrode's(Sigma, T-1788) 제조법은 Embryo tested된 완제품으로 미소적(25㎕)을 만들어 오일도포 후 사용한다.Acid Tyrode's (Sigma, T-1788) manufacturing method is Embryo-tested finished product (25µl) is used after oil coating.
제3단계 : 미분간섭 현미경 작업용 디쉬 제작Step 3: preparing a dish for undifferentiated microscope work
미분간섭(Differential Interference Contrast, DIC)현미경 작업용 디쉬는 다음과 같이 제작하였다.Differential Interference Contrast (DIC) microscope work dishes were prepared as follows.
50mm 디쉬(Falcon 1006) 뚜껑에 직경 1∼3cm로 천공한 다음 Silicon (ShinEtsu KE45)을 디쉬 내부의 구멍주위에 도포한 후, 가로×세로 크기가 2∼4cm의 커버글라스를 실리콘위에 놓고 눌러서 부착시킨다.Perforate the 50mm dish (Falcon 1006) with a diameter of 1 ~ 3cm, apply Silicon (ShinEtsu KE45) around the hole inside the dish, and attach the cover glass of 2 × 4cm in width x length on the silicone .
끝으로, 알코올로 세정한다.Finally, rinse with alcohol.
제4단계 : DIC 현미경용 미소적 제작Step 4: Microfabrication for DIC Microscope
DIC 현미경용 미소적 제작법은 다음과 같이 제작하였다.The microfabrication method for a DIC microscope was produced as follows.
DIC용 작업용 디쉬의 커버글라스 위에 20㎕ 피펫을 이용하여 5∼10㎕ 미소적을 세로로 분주한 다음(미소적의 개수는 난자 20∼30개당 1개씩 계산) 1㎖ 피펫을 이용하여 4㎖정도의 미네랄 오일(sigma, M-3516)을 미소적 주위에 도포한다. 이때, 각 과정에 적합한 배지로 미소적을 형성시킨다.Dispense 5-10 μl micro dropwise vertically using a 20 μl pipette on the cover glass of the DIC working dish (the number of micro drop counts 1 per 20-30 eggs) and then use a 1 ml pipette Oil (sigma, M-3516) is applied around the microspheres. At this time, microdrops are formed with a medium suitable for each process.
제5단계 : 공여핵원용 체세포 준비Step 5: Prepare Somatic Cells for Donor Nuclei
공여핵원용 체세포(Somatic cell line for donor)의 준비는 다음과 같이 수행하였다.Somatic cell line for donor preparation was performed as follows.
Tissue에서 세포주를 분리한다(다음 실험예 참조).Isolate the cell line from the tissue (see the following experimental example).
실험예 1 : 자궁에서의 세포주 분리Experimental Example 1 Isolation of Cell Lines from the Uterus
자궁관류액으로부터의 세포 채취 과장은 먼저, 자궁관류액을 원심분리 시험관에 옮긴 후, 인산염 완충 식염수(PBS) (GIBCO BRL, Life Technologies, USA)에 0.5% P/S항생제(penicillin 10000IU, streptomycin 10mg)를 첨가하여 1500rpm으로 2분간 원심분리한다.Collection of Cells from Uterine Perfusate First, the uterine perfusate was transferred to a centrifugation test tube, followed by 0.5% P / S antibiotic (penicillin 10000IU, streptomycin 10mg) in phosphate buffered saline (PBS) (GIBCO BRL, Life Technologies, USA). ) And centrifuge at 1500 rpm for 2 minutes.
다음, 원심분리 후, 상층액을 버리고 다시 상기의 PBS를 첨가한 후, 피펫을 사용하여 침전된 세포를 재부유시킨다.Next, after centrifugation, the supernatant is discarded and PBS is added again, and the precipitated cells are resuspended using a pipette.
재부유한 세포를 다시 1500rpm으로 2분간 원심분리하며, 이 과정을 3∼5회 반복한다.The resuspended cells are centrifuged again at 1500 rpm for 2 minutes and this process is repeated 3 to 5 times.
최종 원심 세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유하여 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2, 포화습도 배양기내에서 배양한다. 세포가 배양접시 저면에 부착하여 분열 증식하여 단층(monolayer)을 형성하면 이를 1차 세포주(primary cell line)의 성립으로 간주한다.After the final centrifugal rinse, the precipitated cells were suspended in Dulbecco's modified Eagles medium, GIBCO BRL, Life Technologies, USA (DMEM) with 10% FCS, 1% non-essential amino acid solution, and transferred to a cell culture petri dish at 39 ° C. Incubate in 5% CO 2 , saturated humidity incubator. When cells attach to the bottom of the culture plate and divide and proliferate to form a monolayer, this is regarded as the establishment of a primary cell line.
자궁내막에서의 세포 채취 과정은The process of cell collection from the endometrium
먼저, 도축장에서 자궁을 채취하여 청결한 상태로 실험실로 운반한다.First, the uterus is taken from the slaughterhouse and transported to the laboratory in a clean state.
다음, 자궁난관 접합부에서 10cm되는 부위를 멸균된 기구로 횡단한다.Next, 10 cm from the uterine fallopian junction is traversed with a sterile instrument.
횡단면을 집게로 보정한 후, 가위로 3cm 정도 횡단면에서 난관쪽으로 수직 절개한다.After correcting the cross section with forceps, make a vertical incision into the fallopian tube in the cross section about 3 cm with scissors.
절개 후, 수술용 날, 혹은 스크레이퍼(scraper) 등으로 자궁내막을 긁어낸다.After the incision, the endometrium is scraped off with a surgical blade or scraper.
그리고, 채취된 내막의 일부를 1)의 PBS에서 2∼3회 세정 후 0.25% 트립신, 1 mM EDTA 용액(GIBCO BRL, Life Technologies, USA)으로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 30분 정치시킨다. 30분간 정치시킨 후, 와동(vortexing) 혹은 피펫을 사용하여 하여 자궁관류액으로부터의 세포 채취과정과 같은 조건하에 원심분리시킨다.Then, a part of the collected inner membrane was washed 2-3 times in 1) PBS, and then transferred to 0.25% trypsin and 1 mM EDTA solution (GIBCO BRL, Life Technologies, USA) in a saturated humidity incubator at 39 ° C. and 5% CO 2 . Let stand for 30 minutes. After standing for 30 minutes, centrifugation is carried out under the same conditions as the procedure for collecting cells from uterine perfusate using vortexing or a pipette.
상층액을 버리고 침전된 조직 및 세포에 상기 자궁관류액으로부터의 세포 채취과정의 PBS를 첨가하여 재부유시킨 후 다시 원심분리한다.The supernatant is discarded and resuspended by adding PBS from the uterine perfusate to the precipitated tissues and cells, followed by centrifugation.
이 과정을 3∼5회 반복한다.Repeat this process three to five times.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in Dulbecco's modified Eagles medium (GIBCO BRL, Life Technologies, USA) with 10% FCS, 1% non-essential amino acid solution, P / S, and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
세포가 배양접시 바닥에 부착하여 분열 증식하여 단층(monolayer)을 형성하면 이를 1차 세포주의 성립으로 간주한다.When cells attach to the bottom of the culture plate and divide and proliferate to form a monolayer, this is regarded as the establishment of a primary cell line.
실험예 2 : 난관에서의 세포 분리Experimental Example 2 Cell Separation from Fallopian Tube
도축장에서 낭종 등이 없는 정상적인 난소를 선별하여 난관채와 자궁난관 결합부를 포함한 난관을 자궁간막에서 절제하여 청결한 상태로 실험실로 운반한 다음 상기 실험예 1의 PBS 세정 후에, 난관 주변조직을 가위 등으로 절제한다.In the slaughterhouse, normal ovaries without cysts are screened, and the oviducts, including oviducts and ovarian oviducts, are excised from the uterine mesentery and transported to the laboratory in a clean state. Abstinence
다음 멸균된 슬라이드 글라스를 준비하고, 난관채에 인접한 부위를 0.5cm절제 후, 멸균된 슬라이드 글라스위에 고정시킨다.Next, sterilized slide glass is prepared, and the area adjacent to the tubular tube is removed by 0.5 cm and then fixed on the sterilized slide glass.
여분의 슬라이드 글라스의 날을 세워 난관의 중간부위에서 난관채쪽으로 밀어낸다.Raise an extra slide glass blade and push it from the middle of the fallopian tube towards the fallopian tube.
밀려나온 난관상피 조직과 세포를 슬라이드 글라스에서 원심분리용 시험관으로 옮기고, 상기 실험예 1의 PBS를 첨가하여 부유한 후 1의 조건에서 원심분리한다.The extruded fallopian tube epithelial tissue and cells are transferred from the slide glass to the test tube for centrifugation, suspended by adding PBS of Experimental Example 1, and then centrifuged under the conditions of 1.
상기 실험예 1과 같은 방법으로 원심 세정한다.Centrifugal cleaning was performed in the same manner as in Experimental Example 1.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in Dulbecco's modified Eagles medium (GIBCO BRL, Life Technologies, USA) with 10% FCS, 1% non-essential amino acid solution, P / S, and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
세포가 배양접시 바닥 저면에 부착하여 분열 증식하여 단층을 형성하면 이를 1차 세포주의 성립으로 간주한다.When cells adhere to the bottom of the culture dish and divide and proliferate to form a monolayer, this is regarded as the establishment of the primary cell line.
실험예 3 : 난구세포(Cumulus cell)의 분리Experimental Example 3 Isolation of Cumulus Cells
먼저, 난구난자세포 복합체(Cumulus-oocyte complex)를 하이알루로니데이즈(Hyaluronidase)로 처리하여 난자를 제외한 난구세포층을 분리(체세포 핵이식 참조)한 다음 난구세포층에 상기 실험예 1과 같은 트립신(trypsin)-EDTA용액을 첨가하여 39℃, 5% CO2의 포화습도 배양기내에 30분 내지 1시간 정치시킨다.First, the cumulus-oocyte complex is treated with hyaluronidase to separate the oocyte layer except for the oocyte (see somatic cell nuclear transfer), and then trypsin (trypsin) as described above in the oocyte layer. ) -EDTA solution is added and allowed to stand for 30 minutes to 1 hour in a saturated humidity incubator at 39 ° C. and 5% CO 2 .
트립신-EDTA처리 후 와동(vortexing) 및 피펫팅 후 상기 실험예 1과 같은 조건에서 원심세정한다.After vortexing and pipetting after trypsin-EDTA, centrifugal washing is performed under the same conditions as in Experimental Example 1.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in Dulbecco's modified Eagles medium (GIBCO BRL, Life Technologies, USA) with 10% FCS, 1% non-essential amino acid solution, P / S, and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
세포가 배양접시 바닥 저면에 부착하여 분열 증식하여 단층을 형성하면 이를 1차 세포주의 성립으로 간주한다.When cells adhere to the bottom of the culture dish and divide and proliferate to form a monolayer, this is regarded as the establishment of the primary cell line.
실험예 4 : 귀(ear)에서의 세포 분리Experimental Example 4: Isolation of Cells from the Ear
성체 또는 어린 동물의 귀 선단에서 떼어낼 부위 주변의 체모를 깎고, 소독용 알코올과 베타딘으로 소독한다. 멸균된 기구로 1∼2cm2넓이의 피부 조직을 절제한 다음, 50 ml 시험관에 상기 실험예 1의 PBS용액을 준비하여 절제된 조직을 넣어 4℃로 냉장 보관하여 실험실로 운반한다.Shave the hair around the area to be removed from the ear tip of an adult or young animal and disinfect with rubbing alcohol and betadine. After dissecting the skin tissue of 1-2 cm 2 with a sterilized apparatus, prepare the PBS solution of Experimental Example 1 in a 50 ml test tube, and put the excised tissue refrigerated at 4 ℃ and transported to the laboratory.
다시 상기 실험예 1의 PBS 용액으로 세정한 다음, 멸균된 집게로 고정한 후, 체모를 포함한 피부와 연골조직을 멸균된 가위, 날(blade)로 분리하여, 연골조직과 면한 피부 내측의 조직을 채취한다.After washing again with the PBS solution of Experimental Example 1, and fixed with sterile forceps, the skin and cartilage tissue including the hair is separated by sterile scissors and blades, and the tissue inside the skin facing the cartilage tissue is collected. do.
조직편을 다시 상기 실험예 1의 PBS 용액으로 세정 후 칼날(blade)로 세절한 다음, 상기 실험예 1의 0.25% 트립신, 1mM EDTA 용액과 0.8% 콜라게나제형(Collagenase type III) II(GIBCO BRL, Life Technologies, USA) 용액의 혼합액을 첨가하여 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.The tissue pieces were washed again with the PBS solution of Experimental Example 1, and then cut into blades. Then, 0.25% trypsin, 1mM EDTA solution and 0.8% Collagenase type III (GIBCO BRL, Life Technologies, USA) solution is added and incubated in a saturated humidity incubator at 39 ° C., 5% CO 2 .
정치 후 원심 분리용 시험관에 옮겨 와동(vortexing)과 피펫작업 (pipetting) 후, 상기 실험예 1)의 PBS 용액을 첨가하여 같은 조건으로 원심세정한다.After standing, the solution was transferred to a centrifuge test tube, and after vortexing and pipetting, PBS solution of Experimental Example 1) was added thereto and centrifuged under the same conditions.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in 10% FCS, 1% non-essential amino acid solution, P / S added DMEM (Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA) and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
세포가 배양접시 바닥면에 부착하여 분열 증식하여 단층이 형성되면 이를 1차 세포주의 성립으로 간주한다.When cells adhere to the bottom of the culture plate and divide and proliferate to form a monolayer, this is regarded as the establishment of a primary cell line.
실험예 5 : 근육에서의 세포 분리Experimental Example 5: Cell Separation from Muscle
생검(biopsy)에 의해 혹은 신선 사체 등에서 채취한 골격 및 동체의 근육 조직을 1 cm3크기로 절제하여 실험예 4와 같은 방법으로 실험실로 운반한 다음, 조직편을 다시 상기 실험예 1의 PBS 용액으로 세정한 후, 멸균 가위와 칼날(blade)로 세절한다.Muscle tissue of the skeleton and body obtained by biopsy or from fresh carcasses was excised to a size of 1 cm 3 and transported to a laboratory in the same manner as in Experiment 4, and then the tissue pieces were returned to the PBS solution of Experiment 1 After washing, rinse with sterile scissors and blade.
상기 실험예 1의 0.25% 트립신, 1mM EDTA 용액과 0.8% 콜라게나제형 II(GIBCO BRL, Life Technologies, USA) 용액의 혼합액을 첨가하여 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.A mixture of 0.25% trypsin, 1 mM EDTA solution and 0.8% collagenase type II (GIBCO BRL, Life Technologies, USA) solution of Experimental Example 1 was added thereto and cultured in a saturated humidity incubator at 39 ° C. and 5% CO 2 .
정치 후 원심 분리용 시험관에 옮겨 와동(vortexing)과 피펫작업 (pipetting) 후, 상기 실험예 1의 PBS 용액을 첨가하여 같은 조건으로 원심세정한다.After standing, the solution was transferred to a centrifuge test tube, and after vortexing and pipetting, PBS solution of Experimental Example 1 was added and centrifuged under the same conditions.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in 10% FCS, 1% non-essential amino acid solution, P / S added DMEM (Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA) and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
세포가 배양접시 바닥면에 부착하여 분열 증식하여 단층이 형성되면 이를 1차 세포주의 성립으로 간주한다.When cells adhere to the bottom of the culture plate and divide and proliferate to form a monolayer, this is regarded as the establishment of a primary cell line.
실험예 6 : 태아섬유아세포(fetal fibroblast)의 분리Experimental Example 6: Isolation of fetal fibroblasts
먼저, 임신자궁에서 무균적으로 임신 30 내지 60일령의 태아를 채취한다.First, a fetus of 30 to 60 days of gestation is collected aseptically from the uterine uterus.
양막을 포함한 태아를 상기 실험예 4와 같은 방법으로 실험실로 운반한다. 상기 실험예 1의 PBS 용액으로 수차례 세정한다.The fetus including the amniotic membrane is transferred to a laboratory in the same manner as in Experiment 4. It is washed several times with the PBS solution of Experimental Example 1.
두부와 내장 부위를 제거하고 동체와 사지부위에서 연골 조직을 포함하지 않는 부위의 조직을 채취, 멸균 가위와 칼날(blade)로 세절한다.Remove the head and visceral area, remove tissue from the body and limbs that do not contain cartilage tissue, and cut it with sterile scissors and blades.
상기 실험 예 1의 0.25% 트립신, 1mM EDTA 용액과 0.8% 콜라게나제형 II(GIBCO BRL, Life Technologies, USA) 용액의 혼합액을 첨가하여 39℃, 5% CO2의 포화습도 배양기내에서 배양한다. 정치후 원심 분리용 시험관에 옮겨 와동(vortexing)과 피펫작업 (pipetting) 후, 상기 실험예 1의 PBS 용액을 첨가하여 같은 조건으로 원심세정한다.A mixture of 0.25% trypsin, 1 mM EDTA solution and 0.8% collagenase type II (GIBCO BRL, Life Technologies, USA) solution of Experimental Example 1 was added thereto and cultured in a saturated humidity incubator at 39 ° C. and 5% CO 2 . After standing, the sample was transferred to a test tube for centrifugation, and after vortexing and pipetting, PBS solution of Experimental Example 1 was added and centrifuged under the same conditions.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in 10% FCS, 1% non-essential amino acid solution, P / S added DMEM (Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA) and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
세포가 배양접시 바닥면에 부착하여 분열 증식하여 단층이 형성되면 이를 1차 세포주의 성립으로 간주한다.When cells adhere to the bottom of the culture plate and divide and proliferate to form a monolayer, this is regarded as the establishment of a primary cell line.
이하, 분리된 세포주의 유지를 위하여 다음과 같이 조치하였다.Hereinafter, the following measures were taken to maintain the separated cell lines.
실험예 7 : 계대 배양Experimental Example 7: Passage culture
단층이 형성된 1차 세포주는 계대배양을 다음과 같이 실시한다.Primary cell lines with monolayers are passaged as follows.
페트리디쉬의 배양액을 버리고, 상기 실험예 1의 0.25% 트립신, 1mM EDTA 용액을 첨가하여 1 내지 2분 정치 후, 배양용 배지로 교체한다.The culture medium of Petri dish was discarded and 0.25% trypsin and 1 mM EDTA solution of Experimental Example 1 were added thereto, and after 1 to 2 minutes, the culture medium was replaced.
피펫팅하여 세포를 배양 배지 중에 재부유시킨 다음 적당량을 새로운 페트리디쉬에 분주한 후, 배지를 첨가하여 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.The cells are resuspended in culture medium by pipetting, then aliquoted into fresh Petri dishes, and then cultured in a 39 ° C., 5% CO 2 saturated humidity incubator.
실험예 8 : 혈청기아(serum starvation) 배양Experimental Example 8: Serum Starvation Culture
상기 실험예 1의 10% FCS 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's Modified Eagles Medium, GIBCO BRL, Life Technologies, USA)에서 배양, 증식중인 세포주을 0.5 % FCS 첨가 배양액으로 교체한다. 3 내지 10일간 배양 후 공여핵원으로 제공한다.The 10% FCS 1% non-essential amino acid solution of Experimental Example 1, P / S addition cultured in DMEM (Dulbecco's Modified Eagles Medium, GIBCO BRL, Life Technologies, USA), the cell line in proliferation is replaced with 0.5% FCS addition culture. After 3 to 10 days incubation to provide a donor nucleus.
실험예 9 : 세포주의 동결보존Experimental Example 9: Cryopreservation of Cell Lines
유착상태의 세포를 상기 실험예와 같은 방법으로 재부유하여 세포 수가 1 X 105/ml로 되도록 조절한 다음, 세포를 포함한 전체 배양액 중에 DMSO(Dimethyl sulfoxide, Sigma, USA)가 10% 농도가 되도록 첨가하여 희석한 후 Cryovial(Corning, USA)에 봉입한다.Resuspend the adherent cells in the same manner as in Experimental Example to adjust the number of cells to 1 X 10 5 / ml, and then to make 10% concentration of DMSO (Dimethyl sulfoxide, Sigma, USA) in the total culture solution containing the cells. After dilution, the solution is enclosed in Cryovial (Corning, USA).
Cryovial을 -70℃ 냉동고에 넣어 18∼24시간 보관한 후 액체 질소 탱크에 넣어 보관한다.Store Cryovial in -70 ℃ freezer for 18-24 hours and store in liquid nitrogen tank.
실험예 10 : 동결보존 세포주의 융해Experimental Example 10 Fusion of Cryopreserved Cell Lines
액체질소 탱크에서 Cryovial 을 꺼내어 37℃ 온수에 넣어 융해시키고, 융해된 세포를 원심 분리용 시험관에 넣고 상기 실험예 1의 PBS를 첨가하여 1,500rpm, 2분간 원심 세정한다. 상층액을 버리고 세포 침전물을 조심스럽게 하여 원심 세정을 2 회 반복한다.Cryovial is removed from the liquid nitrogen tank and fused in warm water at 37 ° C., and the fused cells are placed in a test tube for centrifugation and centrifuged at 1500 rpm for 2 minutes by adding PBS of Experimental Example 1. Discard the supernatant and carefully centrifuge twice with careful cell precipitate.
최종 원심세정 후, 침전 세포를 10% FCS, 1% 비필수 아미노산 용액, P/S 첨가 DMEM(Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA)에 부유시킨 후, 세포 배양용 페트리디쉬로 옮겨 39℃, 5% CO2의 포화습도 배양기내에서 배양한다.After final centrifugation, the precipitated cells were suspended in 10% FCS, 1% non-essential amino acid solution, P / S added DMEM (Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA) and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO 2 .
실시예 3 : 체세포 핵이식Example 3 Somatic Cell Nuclear Transplantation
제1단계 : 체외성숙(In vitro maturation)Stage 1: In vitro maturation
상기 실시예 1의 체외성숙에서와 같은 방법으로 수핵란을 채취한다.Hydronuclear eggs are harvested in the same manner as in the in vitro maturation of Example 1.
제2단계 : 난구세포 제거(Denuding)Second Step: Denuding
준비물(Preparation)은 다음과 같다.Preparation is as follows.
세정용 피펫, 제거용 피펫, 하이알루로니데이즈, TCM-W 작업용 미소적 작업과정(Procedure)은 다음과 같다.The cleaning procedure, removal pipette, hyaluronates, and TCM-W work process are as follows.
IVM 16∼22시간의 난자를 세정용 마우스 피펫을 이용하여 TCM-W에 1회 세정한다.IVM 16-22 hours eggs are washed once with TCM-W using a cleaning mouse pipette.
하이알루로니데이즈(No. B-③)용액내에 난자를 분주한다.Dispense the egg into the Hyaluronidase solution (No. B-③).
제거용 피펫으로 난구세포를 벗긴다. TCM-W에 3회정도 세정하고 정치시킨다. 상기 작업은 신속하게 수행하는 것이 바람직하다.Peel the oocytes with a removal pipette. Wash and let stand 3 times in TCM-W. It is desirable to perform the work quickly.
제3단계 : 절개 및 탈핵Step 3: Incision and Denuclearization
준비물(Preparation)은 다음과 같다.Preparation is as follows.
작업용 디쉬(No. C), 작업용 미소적 - 사이토칼라신 B(Cytochalasin B), 피펫 : 고정용 피펫, 절개 및 탈핵용(cutting & Enucleation) 피펫Working dishes (No. C), working micro-cytochalasin B, pipettes: fixation pipettes, cutting & enucleation pipettes
절개 및 탈핵과정(Procedure)은 다음과 같다.Incision and denuclearization process is as follows.
작업용 디쉬를 조작판(manipulator plate)위에 위치하도록 놓는 다음, 피펫을 장착한다.Place the working dish on the manipulator plate and fit the pipette.
조작장치(Manipulator)의 왼쪽 암(arm)에 고정용 피펫을 장착한 후 조작장치의 오른쪽 암(arm)에 절개용 피펫을 장착한다.After the fixing pipette is mounted on the left arm of the manipulator, the incision pipette is mounted on the right arm of the manipulator.
피펫의 방향 및 위치를 예컨대 다음과 같이 조정한다.The direction and position of the pipette is adjusted, for example, as follows.
즉, 고정용 피펫은 9시 방향에 위치시키고 절개용 피펫은 3시 방향에 위치시킨다. 피펫 조정기를 중립에 놓아 피펫이 상하 좌우로 자유롭게 움직일 수 있도록 조정한다. 두 피펫을 작업용 점적내로 상하 유동시키면서 피펫이 디쉬의 테두리에 닿지 않도록 각도를 조정하고 피펫 끝이 미소적의 중앙에 위치시킨다.That is, the fixing pipette is positioned at 9 o'clock and the cutting pipette is positioned at 3 o'clock. Place the pipette adjuster in neutral to adjust the pipette to move freely up, down, left and right. With both pipettes flowing up and down into the working drop, angle the pipette so that it does not touch the edge of the dish and the pipette tip is centered in the micro drop.
세정용 마우스 피펫(내경 200㎛이상)을 이용하여 TCM-W에 있던 난자(oocyte) 10∼30개 정도를 1개 군으로 하여 절개 및 탈핵 후 점적으로 이동시킨다.About 10-30 oocytes in TCM-W were grouped into one group using a cleaning mouse pipette (more than 200 µm in diameter) and then moved dropwise after incision and denucleation.
다음 방식으로 절개를 실시한다:The incision is made in the following way:
즉, 조동나사와 미동나사를 이용하여 난자(oocyte)에 먼저 초점을 맞춘 뒤, 2개의 피펫을 상하로 움직여 초점을 조정한다. 2개의 피펫을 움직여서 파지용 피펫의 12시 방향에 제 1극체(The first polar body)가 위치하도록 한다. 파지용 피펫을 난자(oocyte)의 9시 방향에 밀착시킨 뒤 유압을 걸어 난자(oocyte)를 고정시킨다. 절개 피펫을 1시 방향에서 찔러서 투명대를 통과시킨 뒤 세포질이 다치지 않도록 주의하며 11시 방향으로 관통시킨다. 파지용 피펫에 양압을 걸어 난자를 분리하고 파지용 피펫을 절개 피펫이 통과한 제 1극체 상단부의 투명대에 접촉시키고 두 피펫을 마찰하여 투명대를 절개한다.That is, focusing on oocytes first using coarse and fine screws, and then moving the two pipettes up and down to adjust the focus. The first polar body is positioned at 12 o'clock of the gripping pipette by moving the two pipettes. The phage pipette is in close contact with the oocyte at 9 o'clock and hydraulically fixed to the oocyte. Puncture the incision pipette at 1 o'clock, and pass through the zona pellucida. Positive pressure is applied to the gripping pipette to separate the oocytes, and the gripping pipette is contacted with the transparent zone of the upper end of the first polar body passing through the incision pipette, and the two pipettes are rubbed to cut the transparent zone.
탈핵(Enucleation; Squeezing method)은 다음과 같이 수행한다.Nucleation (Squeezing method) is performed as follows.
절개한 난자를 회전시켜 절개창을 수직으로 위치시킨 후 고정용 피펫을 난자의 밑 부위에 위치시켜 난자가 아래쪽으로 움직이지 못하도록 받친 뒤 절개 피펫을 난자의 위에서 눌러 제 1극체를 포함하여 세포질을 10∼30% 탈핵을 실시한다.Rotate the injured egg to position the incision vertically and place the fixation pipette at the bottom of the egg to prevent the egg from moving downward. Then, press the incision pipette on the egg and press down the cytoplasm, including the first polar body. 30% denuclearization.
또는 난자의 절개창을 3시방향에서 수직으로 회전시킨 후 고정용 피펫과 절개 피펫을 아래 위에서 눌러서 제 1극체를 포함하여 세포질의 10∼30% 탈핵을 실시한다. 탈핵시킨 1군의 난자는 TCM-W로 3회 세정하고 핵이식 전까지 IVM용 TCM 199(B-①)에 정치시킨다. 탈핵 후의 난자는 매우 취약하기 때문에 마우스 피펫은 최소한 내경이 300㎛이상 되는 것을 사용하여 작업후 난자의 세포질이 절개창을 통해 빠져 나오지 않도록 주의한다.Alternatively, the incision of the egg is rotated vertically in the 3 o'clock direction, and then the fixation pipette and the incision pipette are pressed down from above to perform 10-30% denucleation of the cytoplasm including the first polar body. The denuclearized group 1 eggs were washed three times with TCM-W and allowed to stand in TCM 199 (B-①) for IVM until nuclear transfer. Since the egg after denuclearization is very fragile, use a mouse pipette with an inner diameter of at least 300 µm to ensure that the cytoplasm of the egg does not escape through the incision after the operation.
제4단계 : 공여핵원용 단일 세포의 분리Step 4: Isolation of Single Cells for Donor Nuclei
페트리 디쉬내의 배양액을 버리고 상기 실험예 1의 0.25% 트립신, 1mM EDTA 용액을 첨가하여 1∼2분 정치시킨다.Discard the culture solution in Petri dishes and leave for 1 to 2 minutes by adding 0.25% trypsin, 1 mM EDTA solution of Experimental Example 1.
정치 후, 1% FCS 첨가 PBS(-)(GIBCO BRL, Life Technologies, USA)에 피펫팅에 의해 재부유시킨다.After standing, resuspended by pipetting in PBS (-) (GIBCO BRL, Life Technologies, USA) with 1% FCS.
세포의 수가 적당량이 되도록 조절하여 (2,000/0.1ml) Eppendorf 시험관에 분주시킨다.Adjust the number of cells to an appropriate amount (2,000 / 0.1 ml) and dispense into Eppendorf test tubes.
제5단계 : 공여세포 이식(Injection)Step 5: Injection of Donor Cells
준비물(Preparation)은 다음과 같다.Preparation is as follows.
작업용 디쉬, 작업용 점적 만들기-PAH, 피펫 : 고정용 피펫, 주사용 피펫, 공여 세포 미소적(Donor 세포 준비 참조)Working dish, working drop making-PAH, pipette: fixation pipette, injection pipette, donor cell micro (see Donor cell preparation)
작업과정(Procedure)은 다음과 같다.The procedure is as follows.
작업용 디쉬를 조작판(manipulator plate)에 위치시킨다.Place the working dish on the manipulator plate.
절개 피펫을 주사용 피펫으로 교체시킨다.The incision pipette is replaced with an injection pipette.
IVM 배지내에 있는 난자(oocyte)를 세정용 마우스 피펫을 이용하여 TCM-W 점적에서 1∼3회 세정후 주입용 미소적으로 이동시킨다.Oocytes in IVM medium are transferred microscopically for injection after one to three washes in a TCM-W drop using a cleaning mouse pipette.
이식용(Injection) 피펫을 공여세포 미소적에 넣고 20∼40㎛크기의 1개 이상의 공여세포를 흡입한 뒤 피펫을 주입용 미소적으로 이동시킨다.Insert the injection pipette into the donor cell microparticles, inhale one or more donor cells of 20-40 μm in size, and then move the pipette microscopically for injection.
절개창을 양 피펫과 1시방향에 수직으로 놓고 고정용 피펫으로 고정한다.Place the incision perpendicular to both pipettes and the 1 o'clock position and secure with a fixed pipette.
이식용(Injection) 피펫을 절개창으로 주입하고, 유압을 걸어 1개의 공여세포을 주입시킨다.An injection pipette is injected into the incision and hydraulically infused one donor cell.
작성된 핵이식란 군을 마우스 피펫으로 세정용 미소적으로 옮겨 1∼3회 세정하고 정치시킨다.The resulting nuclear transfer egg group is transferred to a washing microscopically with a mouse pipette, and then washed and left to set once or three times.
실시예 4 : 세포융합(Cell Fusion)Example 4 Cell Fusion
세포융합에 필요한 준비물(Preparation)은 기기-BTX ECM 2001의 다음과 같다.Preparations required for cell fusion are as follows in Instrument-BTX ECM 2001.
만니톨(Manitol)을 4-웰 디쉬의 3개의 웰(1번, 2번, 3번 웰, 순서는 상관없음)에 0.4ml∼1ml 분주하고, 나머지 1개의 웰 (4번)에는 10% TCM-W가 첨가된 만니톨을 제조한다.Manitol is dispensed from 0.4 ml to 1 ml into three wells of 4-well dishes (wells 1, 2 and 3, in any order) and 10% TCM- for the other well (4). Prepare mannitol to which W is added.
만일 융합과 활성화를 동시에 실시할 경우는 Ca2+첨가 융합배지를 사용하며, 융합후 활성화군에서는 Ca2+를 배제한 융합배지를 사용한다.If fusion and activation are performed at the same time, Ca 2+ -added fusion medium is used, and in the activation group after fusion, Ca 2+ fusion medium is used.
다음, 2개 전극(3.2mm chamber No. 453)에 만니톨을 0.2∼1ml 넣고 BTX-세포 조작기와 연결시킨다.Next, 0.2-1 ml of mannitol is placed in two electrodes (3.2 mm chamber No. 453) and connected to a BTX-cell manipulator.
이때, 세포융합 조건은 다음과 같다.At this time, the cell fusion conditions are as follows.
DC 조건 : 전압(Voltage)은 0,75∼2.25kv/cm이며, 횟수(Times)는 1∼2회, 시간(Seconds)은 회수×시간 총 60㎲ 이내로 조정한다.DC condition: Voltage is 0,75 ~ 2.25kv / cm, Times is 1 ~ 2 times, and Seconds is adjusted within 60ms of recovery time × time.
[태아섬유아세포(Fetal fibroblast) - DC 1.75∼1.85KV/cm, 20㎲, 1회][Fetal fibroblast-DC 1.75-1.85 KV / cm, 20 Hz, 1 time]
(Ear 세포 - DC 1.75∼1.8KV/cm, 15㎲, 1sec 간격, 2회)(Ear cell-DC 1.75 ~ 1.8KV / cm, 15㎲, 1sec interval, 2 times)
[자궁(Uterine) 세포 - 1.85∼2.25KV/cm, 15㎲, 1sec 간격, 2회][Uterine Cells-1.85-2.25 KV / cm, 15 Hz, 1 sec interval, twice]
작업과정(Procedure)은 다음과 같다.The procedure is as follows.
작업 1시간전 BTX를 가동하여 예열한 다음 세정용 마우스 피펫에 10∼20㎕의 만니톨을 빨아들인 후 핵이식란이 있는 TCM-W 미소적에 첨가한다.One hour prior to operation, the BTX was run and preheated, and 10-20 μl of mannitol was sucked into the cleaning mouse pipette and added to the microtransplanted TCM-W microspheres.
1분 이상 정치시킨 후 세정용 마우스 피펫을 이용하여 4번 웰(10% TCM-W 만니톨)에 핵이식란을 주입한다.After standing for at least 1 minute, a nuclear transfer egg is injected into well 4 (10% TCM-W mannitol) using a cleaning mouse pipette.
1분 이상 정치시킨 후 세정용 마우스 피펫을 이용하여 1번 웰에 넣고, 같은 방법으로 2번 웰에 핵이식란을 이동시킨다.After standing for 1 minute or more, the wells are placed in the well 1 using a cleaning mouse pipette, and the nuclear transfer egg is transferred to the second well in the same manner.
마우스 피펫을 이용하여 핵이식란을 하나씩 2개 전극 사이에 놓고 공여세포가 (+)전극을 향하도록 정렬시킨다.Using a mouse pipette, the nuclear transfer eggs are placed between two electrodes one by one and aligned so that donor cells face the positive electrode.
통전을 실시하고 융합한 핵이식란은 3번 웰에 모아서 1개 군의 융합이 끝나면 TCM-W로 옮겨 3회 세정한다.The energized and fused nuclear transfer embryos are collected in three wells and transferred to TCM-W for cleaning three times after the fusion of one group is completed.
Cytochalasin B 미소적내에서 30∼60분간 처리후, 융합·활성을 동시에 실시하게 될 군은 후활성화(postactivation) 과정으로, 융합 후에 활성화시킬 군은 상기의 화학물질을 이용하여 활성화시킨다.After treatment for 30 to 60 minutes in the cytochalasin B microsphere, the group that will perform the fusion and activity at the same time is a postactivation process, and the group to be activated after the fusion is activated using the above chemicals.
활성화(Activation)는 다음과 같이 실시한다.Activation is performed as follows.
융합과 동시에 일렉트로퓨젼(elecrofusion) 방법으로 활성화를 유도하는 경우에는 Ca2+ 첨가 융합배지내에서 일렉트로퓨젼(electrofusion)을 실시한다.In the case of induction of activation by the electrofusion method at the same time as the fusion, electrofusion is performed in a Ca 2+ -added fusion medium.
Ionomycin(B-⑨)처리법으로 활성화를 유도하는 경우에는, B-⑨, 50㎕ 점적내에서 4분간 정치(빛에 민감하므로 되도록 빛의 유입을 차단)하고, 35mm 디쉬내 10% FBS 혹은 30mg/ml BSA 첨가하고 세정용 매질하에서 이노마이신 (Ionomycin)을 제거한 뒤 5분간 정치시킨다.In the case of induction of activation by the ionomycin (B-⑨) treatment, B-⑨, incubated for 4 minutes in a 50 µl drop (it is sensitive to the light, blocking the inflow of light as much as possible), and 10% FBS or 30 mg / in 35 mm dish. ml BSA is added and the inomycin is removed under washing medium and left to stand for 5 minutes.
실시예 5 : 융합 후 활성화(Postactivation)Example 5 Postactivation
Cycloheximide 25㎕ 미소적 내에 5∼10개 정도의 작성란을 침지하여 6시간 배양한다.Soak 5-10 eggs in 25µl of cycloheximide and incubate for 6 hours.
DMAP 25㎕ 미소적 내에 5∼10개 정도의 작성란을 침지하여 4시간 배양시킨다.Five to ten eggs are immersed in 25 µl of DMAP and incubated for 4 hours.
배양은 다음과 같이 한다.Incubate as follows.
상기 실시예 2 제2단계의 배양액(Culture medium) 중 택일하여 5% CO2배양기 또는 5% CO2, 7% O2, 88% N2에서 5∼8일간 배양한다. 활성화(Activation) 후 24시간 전후로 검란, 발육단계별로 구분하여 미소적내에 배치한다.Example 2 The culture medium of the second step (Culture medium) is alternatively incubated for 5-8 days in a 5% CO 2 incubator or 5% CO 2 , 7% O 2 , 88% N 2 . After about 24 hours after activation (Activation), it is arranged within the microscopic division by egg phase and development stage.
최종 검란은 수정 7일에 실시하여 후기배 발육율을 계산하고 I-4, 5의 방법으로 핵이식란의 동결 및 이식을 실시한다.The final eggs should be fertilized on the 7th day of fertilization to calculate the late embryonic development rate and the freezing and transplantation of the nuclear transfer embryos using I-4 and 5.
이상과 같이, 소의 체외수정 및 체외성숙에 의해 얻어진 수정란 또는 소의 체세포를 채취하여 세정 및 분주처리조건하에 보관하고, 소의 다양한 조직으로부터 세포주를 분리보존하고, 최초 세포의 세포막의 일부를 절개하고 세포질의 10 내지 30%의 탈핵을 실시한 다음, 상기 공여세포를 상기 핵이 탈핵된 난자에 이식하여 전기융합시키고 활성화 및 후활성화 단계 후, 배양 및 성숙시킴으로써, 체세포의 복제에 의해 소를 생산할 수 있게 된다.As described above, the fertilized egg or bovine somatic cells obtained by bovine in vitro fertilization and in vitro maturation are collected and stored under the conditions of washing and dispensing, and the cell line is separated and preserved from various tissues of the bovine, and a part of the cell membrane of the first cell is cut and cytoplasmic After 10-30% of denuclearization, the donor cells are transplanted into the nucleated denuclearized egg, followed by electrofusion, followed by culturing and maturation after the activation and post-activation steps, so that cows can be produced by replication of somatic cells.
이하에서는 전술한 본 발명의 각각의 공정에 사용되는 다양한 배지들의 종류와 그 조성을 제시한다:The following describes the types and compositions of the various media used in each of the processes of the present invention described above:
소 항생제(bovine antibiotics)는 다음과 같다.Bovine antibiotics include:
P/S bottle(Sigma, Penicillin 10000IU, Streptomycin 10mg)에 20ml DW를 첨가Add 20ml DW to P / S bottle (Sigma, Penicillin 10000IU, Streptomycin 10mg)
FSH(Antrin, 일본 Denka)/ Estradiol(Sigma, E-8875)은 다음과 같다.FSH (Antrin, Japan Denka) / Estradiol (Sigma, E-8875) are as follows.
1. TCM-C 5㎖를 1 vial에 넣고(10㎎/5㎖)1.Add 5ml of TCM-C to 1 vial (10mg / 5ml)
2. 1㎖를 취한다. -(2㎎을 취한 셈)2. Take 1 ml. -(Takes 2 mg)
3. 딴 1㎖를 TCM-C 9㎖와 합친다.3. Combine the other 1 ml with 9 ml TCM-C.
(2㎖/10㎖) ⇒ 200㎕씩 eppendorf 시험관에 분주하여 냉동보관(2mL / 10mL) ⇒ Aliquot 200µl into eppendorf test tube
Estradiol : 1㎍/ml의 농도로 맞추려면 10ml의 에탄올에 5mg의 estradiol을 희석하고 1㎕를 0.5ml의 4-웰 디쉬에 첨가하면 된다. 이 때 에탄올이 증발하도록 빈 디쉬의 바닥에 estradiol을 먼저 분주하고 증발한 다음 FSH, 배양액등을 분주한다.Estradiol: To adjust the concentration to 1µg / ml, dilute 5mg of estradiol in 10ml of ethanol and add 1µl to 0.5ml of 4-well dish. At this time, estradiol is first dispensed on the bottom of the empty dish so as to evaporate ethanol, and then the FSH and the culture solution are dispensed.
헤파린(200㎕/㎖)은 Cap-TALP 10ml에 0.002g 헤파린(sigma, H-3149)를 첨가하여 eppendorf 시험관에 300㎕씩 분주한후 냉동보관한다.Heparin (200µl / ml) is added to 0.002g heparin (sigma, H-3149) in 10ml of Cap-TALP, and 300µl aliquots are added to eppendorf test tubes.
공배양은 다음과 같이 수행한다.Co-culture is carried out as follows.
도축장에서 낭종 등이 없는 정상적인 난소를 선별하여 난관채와 자궁난관 결합부를 포함한 난관을 자궁간막에서 절제하여 청결한 상태로 실험실로 운반하여At the slaughterhouse, normal ovaries without cysts are screened and the oviducts, including fallopian tubes and uterine fallopian joints, are removed from the mesentery and transported to the laboratory in a clean state.
DPBS로 세정 후, 난관 주변 조직을 가위 등으로 trimming하고 멸균된 슬라이드 글라스를 준비하여 난관채에 인접한 부위를 0.5cm절제 후, 멸균된 슬라이드 글라스위에 고정한 다음 여분의 슬라이드 글라스의 날을 세워 난관의 중간부위에서 난관채 쪽으로 밀어낸다.After washing with DPBS, trimming the tissues around the fallopian tube with scissors and preparing a sterilized slide glass, cutting the area adjacent to the fallopian tube 0.5cm, fixing it on the sterilized slide glass, and then setting the edge of the extra slide glass to raise the middle of the fallopian tube. Push from the site to the fallopian tube.
밀려 나온 난관상피 조직과 세포를 슬라이드 글라스에서 원심분리용 시험관로 옮기고, DPBS를 첨가하여 부유한 후 원심(1500rpm, 2분)분리한다.The extruded fallopian tube epithelial tissue and cells are transferred from the slide glass to the centrifuge test tube, suspended by adding DPBS, and centrifuged (1500 rpm, 2 minutes).
DPBS로 2회 원심세정한 다음, 마지막 세정 후 펠릿을 5000∼10000개/ml의 세포농도가 되도록 TCM 199(No. 3)를 첨가하여 부유한다. 4웰 위의 부유액 500㎕을 분주, 배양하여 confluent monolayer(2×104)를 얻는다.After centrifugation twice with DPBS, the pellet was suspended by adding TCM 199 (No. 3) to a cell concentration of 5000-10000 / ml after the last washing. Dispense and culture 500 µl of suspension on 4 wells to obtain a confluent monolayer (2 × 104).
각 웰당 수정란 25∼50개를 첨가하여 공배양하게 되며 medium change는 2일 간격으로 실시한다.Each well is cocultured with 25 to 50 fertilized eggs and medium changes are carried out every two days.
실시예 6Example 6
한우의 귀에서 채취한 세포를 공여핵세포로 사용하고 1.75kv/cm의 전압을 사용하여 통전시간 및 통화 횟수에 따른 체세포 복제성적을 비교한 결과를 하기 표 11에 나타내었다:Table 11 shows the results of comparing the somatic cell replication results according to the energization time and the number of calls using the cells collected from the ears of Hanwoo as donor nuclear cells and using a voltage of 1.75kv / cm:
한편, 홀스타인종 젖소의 자궁에서 채취한 세포를 공여핵세포로 사용하여 융합키고, 이것을 상기 한우와 비교하였다(표 2).On the other hand, cells harvested from the uterus of Holstein cows were fused using donor nuclei cells, which were compared with the Korean beef (Table 2).
또한, 공여핵세포의 제공원(자궁, 귀)에 따라 융합 후 세포의 발육률을 비교실험한 결과를 표 3에 나타내었다.In addition, Table 3 shows the results of comparing the growth rate of the cells after fusion according to the donor nucleus cells (uterine, ear).
배반포성숙률은 핵이식 복제과정중의 체외배양에서 대리모에 이식하기 전단계까지 발육된 후기 수정란의 상태를 말한다.The blastocyst maturation rate refers to the state of late fertilized eggs developed from in vitro culture during nuclear transfer cloning to pre-transplantation into surrogate mothers.
본 발명의 세포융합방법에 따라 복제 소의 생산이 가능하게 되었고, 이에 따라, 장차 의약학, 축산학 분야에서 복제 소를 이용한 약제 또는 장기 등의 생산 등과 관련된 무한한 응용가능성이 기대되며, 본 발명에 따라 복제 소를 생산하는 기술은 반드시 소에 국한되지 않고 향후의 광범위한 시도에 따라 변형되어 상이한 종의 동물들에도 적용될 수 있을 것이다. 본 발명에 따라 복제 동물을 생산 및 그 응용분야가 개척될 수 있는 새로운 장이 열릴 것으로 예상된다.Production of cloned cows was made possible according to the cell fusion method of the present invention. Accordingly, the present invention is expected to be infinitely applicable in the manufacture of drugs or organs using cloned cows in the fields of pharmacy and animal husbandry, and according to the present invention. The technology for producing larvae is not necessarily limited to cattle, but may be modified in a wide variety of future trials to be applied to animals of different species. It is anticipated that new fields will be opened in which the production of cloned animals and their applications can be pioneered according to the present invention.
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KR100829426B1 (en) * | 2007-01-17 | 2008-05-15 | 재단법인서울대학교산학협력재단 | Method for producing cloned dog |
US9249385B1 (en) | 2014-12-18 | 2016-02-02 | City University Of Hong Kong | System and method for fusing cells |
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KR100829426B1 (en) * | 2007-01-17 | 2008-05-15 | 재단법인서울대학교산학협력재단 | Method for producing cloned dog |
US9249385B1 (en) | 2014-12-18 | 2016-02-02 | City University Of Hong Kong | System and method for fusing cells |
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