J. Exp. Biomed. Sci. 10 (2004) 391 395 Feasibility of Coculture Method for Production of Chimeric Mice Using J1 Embryonic Stem Cells Hye-Jun Shin 1,2, Sung-Sik Park 1, Sun-Uk Kim 1,3, Sang-Mi Cho 1, Ying-Hao Han 1,2, Hyun-Sun Kim 1, Sang-Geun Kim 1,2, Dong-Seok Lee 1 and Dae-Yeul Yu 1 1 Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-806, Korea 2 College of Vaterinary Medicine, Chungnam National University, Daejeon 305-764, Korea 3 Laboratory of Reproductive and Developmental Biology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea The demand for the production of gene-defective mice from embryonic stem (ES) cells is increasing to clarify decisive gene function in vivo. Although blastocyst injection is widely used to generate ES cell-mediated knockout mice, coculture method has been alternatively used because of several advantages, such as low cost and simple procedure. Thus, this experiment was designed to demonstrate the feasibility of the coculture method using J1 ES cells, which are known to be efficient for blastocyst injection. Eight-cell embryos were harvested from 2.5 days post-coitum (dpc), denuded with acid tyrode's solution, and transferred onto trypsinized J1 ES cells. Aggregation was carried out following two typical methods, which are simple coculture method and aggregation in groove prepared by aggregation needle. Successfully aggregated-embryos were developed to blastocysts for 24 h and transferred into uterus of pseudo-pregnant foster mother. Chimeric offspring was judged by coat pigmentation. In this study, we could obtain chimeric mice from all the two aggregation methods, but the chimera production efficiencies in coculture using groove were three times higher at least than those in the other group. In conclusion, these observations suggest that coculture method should be available for production of knockout mice from J1 ES cells. Presently, the germ-line transmission rates of the chimeras produced from the two methods are under investigation. Key Words: Embryonic stem cell, Coculture aggregation, Chimeric mice 서 초기수정란에서의발생과정과유전적조절연구는생쥐배아줄기세포 (embryonic stem cell, ES cell) 의개발과더불어유전자의기능연구에많은가능성과기회를제공하여왔다. 이러한배아줄기세포는배반포의내부세포괴 (inner cell mass, ICM) 로부터유래된것으로서체외에서배양한후에도다능성을유지하는것으로알려져있다 (Evans et al., 1981; Martin, 1981). 이와더불어비교적무제한적으로증식이가능하다는특성때문에삽입된유전자에의한상동적재조합 (homologous recombination) 에의하여특정유전자를결손시키는것 * 논문접수 : 2004 년 11 월 10 일수정재접수 : 2004 년 12 월 10 일 교신저자 : 이동석, ( 우 ) 305-806 대전광역시유성구어은동 52 번지, 한국생명공학연구원인간유전체연구실 Tel: 042-860-4349, Fax: 042-860-4608 e-mail: lee10@mail.kribb.re.kr 론 이가능하였다 (Gossler et al., 1989; Friedric et al., 1991; Robertson, 1987). 이와같은방법을이용하여만들어진유전자결손생쥐들은유전자의기능연구에있어매우효과적으로이용되고있으나많은방법적인복잡성은개선의여지가필요한실정이다. 유전자결손생쥐를효율적으로생산하기위해서는카이미라 (chimeras) 생쥐의생산성을재고하는것이중요하다. 가장보편적인방법으로는미세하게세공된유리바늘을이용하여배반포의포배강에배아줄기세포를직접주입하는배반포미세주입법이있다 (Bradley, 1987). 이방법은생식선이행능을가진카이미라생쥐의생산성이높은것으로보고되어널리이용되고있으나, 많은시간, 노력및숙련을요하는단점이있다. 더욱이이기술을수행하는데에는 pipette puller, microforge, grinder 및 micromanipulator와같은고가의특수한장비들을필요로하기때문에비용면에서도매우제한적이다. 이와는대조적으로투명대를제거한수정란과배아줄기세포를공배양하여응집카이미라를생산 - 391 -
하는기술은비교적적은숙련을요할뿐만아니라비용절감효과를큰장점으로가지고있는것으로알려져있다. 특히많은수의카이미라수정란을생산할수있다는장점은비교적낮은카이미라의생산성을극복할수있을것으로기대되고있다. 따라서본연구에서는공배양방법을통한카이미라생쥐의생산가능성을조사하고유전자결손생쥐생산에가장널리이용되고있는 J1 배아줄기세포의이용성을극대화하고자한다. 재료및방법 1. 8-세포기수정란의준비실험에공시된모든생쥐는온도 22±1, 습도 45±10% 및 12시간동안 (AM 7:00~PM 7:00) 조도가유지된 specific pa- thogen-free/virus antibody-free 환경하에서사육되었다. 6주령의 ICR 암컷생쥐의복강에 5 IU의 pregnant mare's serum gona- dotropin (Folligon, Intervet Co., The Netherlands) 를복강주사하고그로부터 46~48시간후에 5 IU의 human chorionic gona- dotropin (Chorulon, Intervet Co., The Netherlands) 을복강주사하여과배란을유도하였다. 과배란유도된암컷생쥐를동종의수컷생쥐와 1:1로교미시켰으며, 다음날오전에질전 (va- ginal plug) 을확인하여임신여부를판단하였다. 교미일로부터 2.5일후에암컷생쥐를경추탈골로도살하고복강을절개하여난관 (oviduct) 및자궁 (uterus) 을적출하였다. 0.4% BSA가첨가된 M2 배양액 (Quinne et al., 1982) 을난관누두부 (am- pulla) 로부터관류시킴으로써 8-세포기수정란을회수하였으며, acid tyrode's 용액을이용하여투명대를제거한후응집에사용되었다. 2. 배아줄기세포의배양 J1 배아줄기세포의배양은 Dulbecco's modified Eagle's medium (DMEM, Invitrogen) 을기본배양액으로하여 10% fetal bovine serum (FBS, HyClone), 5% ES-qualified FBS (Invitrogen), 0.1 mm β-mercaptoethanol (Sigma-aldrich), 1 nonessential amino acids (Invitrogen), antibiotics (Invitrogen) 및 1000 IU의 leukemia inhibitory factor (Chemicon) 를첨가하여사용하였다. Feeder layer는임신 13.5일령의생쥐태아로부터회수한섬유아세포 (mouse embryonic fibroblast, MEF) 를 10 µg/ml mitomycin C (Sigma-aldrich) 로 3시간처리하여준비하였다. 본실험에서사용된 J1 배아줄기세포는 peroxiredoxin 유전자를결실시키는데사용된세포를 subcloning하여사용하였다. 3. 8-세포기와 ES 세포의공배양응집카이미라의생산은단순공배양 (Woods et al., 1993) 및 aggregation needle을이용한공배양법을이용하였다. 간단히요약하면, log phase에서증식중인 J1 배아줄기세포를 0.25% trypsin/5.3 mm EDTA 를처리하여분리하고, 30분동안 37, 5% CO 2 Incubator에서배양하여상층액을회수함으로써용기바닥에먼저고착된 MEF를제거하였다. 단순공배양법에의한응집카이미라의생산을위해배아줄기세포의농도를 1.5 10 6 /ml로조정한후 60 mm 배양용기 (Nunc) 에 20 µl의미세액적으로제조하였으며, 각미세액적내에약 5~ 10개의투명대가제거된 8-세포기수정란을옮겨 2시간동안공배양하였다 (Fig. 1). 배양후 8-세포기수정란의표면에부착된 ES 세포의수가 5~6개가되도록피펫팅하여조정하였다. 응집이완료된수정란은 0.4% BSA가첨가된 M16 배양액 (Whittingham, 1971) 에넣어 20시간동안 37, 5% CO 2 incu- J1 ES Subclones No. of embryos aggregated Table 1. Production of chimeras by ES cell-embryo coculture No. (%) of blastocysts transferred a No. (%) of No. (%) of pups No. (%) of chimeras pregnants/fm used Born b Survivied c produced d #1 466 332 (71.2) 8/13 (61.5) 22 (6.7) 18 (81.8) 1/18 (5.5) #2 380 268 (70.5) 5/8 (62.5) 18 (6.8) 13 (72.2) 2/13 (15.3) Abbreviations are FM, foster mother. a Some newborn mice died due to cannibalism and/or artifical accidents. b Birth rate=no. of pups born/ no. of blastocysts used. c Survival rate=no. of pups survived/no. of pups born. d Chimera productivity=no. of chimeras/ no. pups survived. J1 ES Subclones No. of embryos aggregated Table 2. Production of aggregation chimeras by aggregation needle No. (%) of blastocysts transferred a - 392 - No. (%) of No. (%) of pups No. (%) of chimeras pregnants/fm used Born b Survivied c produced d #1 359 312 (86.9) 7/12 (58.3) 16 (5.2) 11 (68.7) 4/11 (36.3) #2 413 336 (81.4) 7/13 (53.8) 19 (5.7) 14 (73.6) 5/14 (35.7) Abbreviations are FM, foster mother. a Some newborn mice died due to cannibalism and/or artifical accidents. b Birth rate=no. of pups born/ no. of blastocysts used. c Survival rate=no. of pups survived/no. of pups born. d Chimera productivity=no. of chimeras/ no. pups survived.
A B Fig. 1. ES cell-embryo coculture. (A) Eight-cells (arrowhead) settled on a lawn of J1 ES cell. (B) Compacted eight-cell embryos with single cells or groups of J1 ES cells attached (arrowheads) after a 2 hr coculture. These embryos were moved to M16 droplets for overnight culture to the blastocyst stage. bator에서배양하였다. 또다른응집카이미라의생산방법은 60mm 배양용기의바닥에 darning needle (aggregation needle) (BLS, Budapest, Hungary) 을이용하여홈을만든후, 투명대가제거된 8-세포기수정란을하나씩넣고, 개개의세포로된 ES 세포를 10~15개정도로조정하여넣어준뒤 24시간동안 37, 5% CO 2 incubator에서배양하였다 (Fig. 2). 4. 응집카이미라생쥐의생산배반포로발달시킨카이미라 (chimeras) 수정란은위임신유기 2.5일령의대리모의왼쪽자궁에 15~25개씩이식하였다. 태어난새끼들중모피의색을관찰하여카이미라생쥐의선별및정도를판단하였다. 결과 1. 단순공배양법에의한카이미라생쥐의생산 J1 배아줄기세포 subclone를단순공배양법에의하여카이미라생쥐를생산한결과는 Table 1에정리하였다. 2개의 subclone과의응집에사용된 8-세포기수정란은각각 466 및 380개이며, 이들로부터총 332 및 268개의카이미라배반포를생산하여각각 8 및 5마리의임신된대리모로부터각각 22 (6.7%) 및 18마리 (6.8%) 의산자를얻을수있었다. 대리 모의포유중각각 18 (81.8%) 및 13 (72.2%) 마리의산자가생존하였고, 모피색의관찰결과카이마라의생산효율은각각 5.5 및 15.3% 인것으로조사되었다. 2. Aggregation needle 을이용한응집카이미라생쥐의생산 Aggregation needle을이용하여만든홈에서 24시간동안의공배양결과는 Table 2에정리하였다. 2개의 J1 배아줄기세포 subclone과응집된각각 359 및 413개의 8-세포기수정란으로부터총 312 및 336개의카이미라배반포를생산하였으며, 대리모에이식한후각각 16 (5.2%) 및 19마리 (5.7%) 의산자를얻을수있었다. 대리모의포유중각각 11 (68.7%) 및 14 (73.6%) 마리의산자가생존하였고, 모피색을관찰한결과각각에서 4 (36.3%) 및 5마리 (35.7%) 의카이미라생쥐를얻을수있었다 (Fig. 3). 고찰배아줄기세포에상동적재조합법으로유발시킨유전자결손은생체내의유전자의기능을밝혀내는데있어매우결정적이고효율적인방법이라할수있다 (Gossler et al., 1989; Friedrich et al., 1991). 그러나이러한과정은매우복잡하고많 - 393 -
A B Fig. 2. Embryo ES cell aggregation coculture. (A) Single embryo of eight-cell (arrowheads) and cluster containing 10~15 ES cells were grooved in a hole made by an aggregation needle. (B) The embryo-es cell aggregate cultured with M16 for 24 hr. Fig. 3. Production of chimeras by aggregation. Chimeric mice prepared from coculture of CD1 (albino) embryos with J1 ES cells derived from 129 Sv (agouti) strain of mice. The chimeric mice were mated with CD1 mice at 8 weeks old, finding for the germline transmission. 은비용을요하기때문에방법적인개선을위한다양한연구가있어왔다. 특히, 카이미라수정란을제작하는데있어기존에행해져오던실험방법들의이러한문제점을보다개선하기위하여투명대가제거된초기배와의공배양법이고안되었다. 이방법은배반포에미세주입하는방법에비하여고가의장비나, 특수한기술또는많은숙련을요하지않기때문에유전자조작한생쥐의생산을보다용이하게할수있다 (Gen et al., 1999). 카이미라생쥐의생산효율은생쥐의과 배란유도시의반응이나회수한수정란의상태그리고이식되었을때배아줄기세포와수정란의계통적조화 (strain compatibility) 등여러가지요인들에의해결정되며 (Schuster- Gossler et al., 2001), 본연구에서도출된결과들은국내에서는최초로미세주입에전적으로이용되어온 J1 세포또한간단한공배양법에의해카이미라생쥐를생산할수있음을보여준것이라할수있다. 본연구에서는카이미라생쥐를생산하기위하여공배양응집법을사용하였으며그과정에서도배아줄기세포와수정란의응집과정에있어약간의차이를두어실험하였다. 첫번째단순공배양방법은배아줄기세포를배양용기에먼저배양한후그위에수정란을올려놓아응집을유도한것으로써 (Fig. 1A) 현미경하에서확인한결과응집이성공적으로이루어졌음을확인할수있었다 (Fig. 1B). 또다른방법은배양용기에 aggregation needle 로홈을만들고그안에한개의수정란 (Fig. 2A) 과 1~15개의배아줄기세포를넣어 24시간동안응집을유도한것으로써응집이성공적으로이루어졌음을확인할수있었다 (Fig. 2B). 이두방법모두를통해서로유사한산자출생률및생존율을얻을수있었으나카이미라의생산효율은두번째방법이우수한것으로조사되었다. 이러한결과는수정란의표면에붙은배아줄기세포의숫자, 공배양시간및응집력을증가시키는환경조성의중요성을시사한다할수있다. 또한응집수정란의배반포발달 - 394 -
률에있어단순응집법 (71.2 및 70.5%, Table 1) 에비해두번째방법 (86.9 및 81.4%, Table 2) 이월등히높은것으로조사되어, 여러가지측면에서후자의방법이보다효율적이라판단되었다. 그러나이러한응집배양간결과의차이가심각하게존재한다할지라도본연구에사용된두가지방법모두에서카이미라생쥐를양산할수있었기때문에 J1 배아줄기세포를이용한유전자결손생쥐생산에미세주입과마찬가지로공배양기법또한효과적으로이용될수있을것으로기대되었다. 현재태어난카이미라생쥐의생식선이행능력을검증하기위하여역교배를실시하고있으며, 이와더불어공배양방법의개선을통하여카이미리즘빈도를재고하기위한다각적인방법을모색중에있다. Acknowlegement This work was supported by the 21st Century Frontier program in Functional Human Genome Project of Korea Grant No. HGC030-0324 and a grant from KRIBB Research Initiative program. REFERENCES Bradley A. Production and analysis of chimeric mice. In: Robertson EJ (eds.), Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford. 1987: 113-152. Evans MF, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981. 292: 154-156. Friedric G, Soriano P. Promoter traps in embryonic stem cells: A genetic screen to identify and mutate developmental genes in mice. Genes Dev. 1991. 5: 1513-1523. Gossler A, Joyner AL, Rossant J, Skarnes WC. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science. 1989. 244: 463-465. Gen K, Yoichi Y, Kayo Y, Yutaka S, Soh O, Yuka N, Takashi M, Junji T. Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method. J Biochem Biophys Methods. 1999. 39: 137-142. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA. 1981. 78: 7634-7638. Robertson EJ. Embryo-derived stem cell lines. In: Robertson EJ (eds.), Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford. 1987: 71-112. Schuster-Gossler K, Lee AW, Lerner CP, Parker HJ, Dyer VW, Scott VE, Gossler A. Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. Biotechniques. 2001 31: 1022-1026. Wood SA, Allen ND, Rossant J, Auerbach A, Nagy A. Noninjection methods for the production of embryonic stem cellembryo chimeras. Nature. 1993. 365: 87-89. Wood SA, Pascoe WS, Schmidt C, Kemler R, Evans MJ, Allen ND. Simple and efficient production of embryonic stem cellembryo chimeras by coculture. Proc Natl Acad Sci USA. 1993. 90: 4582-4585. - 395 -