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Efficient Generation of Human IgG1 Light Kappa Constant Region Knock-in Mouse by CRISPR/Cas9 System
Biomed Sci Letters 2019;25:372-380
Published online December 31, 2019;  https://doi.org/10.15616/BSL.2019.25.4.372
© 2019 The Korean Society For Biomedical Laboratory Sciences.

Sundo Jung,*

Department of Biomedical Laboratory Science, Shinhan University, Gyeonggi-do 11644, Korea
Correspondence to: Sundo Jung. Department of Biomedical Laboratory Science, Shinhan University, 95, Hoam-ro, Uijeongbu-si, Gyeonggi-do 11644, Korea.
Tel: +82-31-870-3714, Fax: +82-31-870-3719, e-mail: jungsd93@shinhan.ac.kr
*Professor.
Received November 12, 2019; Revised December 11, 2019; Accepted December 20, 2019.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract

Mice with specific modified genes are useful means of studying development and disease. The CRISPR/Cas9 system is a very powerful and effective tool for generating genetically modified mice in a simple and fast manner. To generate human IgG light kappa constant knock-in mice, we tested by microinjection of a mixture of Cas9 protein, single-guide RNA and target homologous recombinant donor DNA into zygotes. We found that the injection of 10 ng/μL of Cas9 protein and crRNA/tracrRNA, rather than single guide RNA, induced the production of knock-in mice more effectively. Thus, our study provides valuable information that will help to improve the production of knock-in mice and contribute the successful generation of humanized Ab-producing mice in Korea.

Keywords : CRISPR/Cas9 system, Knock-In mouse, Human IgG light kappa constant region, Gene editing
꽌 濡

留덉슦뒪뒗 룷쑀瑜 紐⑤뜽 룞臾쇰줈 쑀쟾옄 蹂삎쓣 넻빐 쑀쟾옄쓽 湲곕뒫쓣 씠빐븯怨 吏덈퀝 移섎즺 뿰援ъ뿉 愿묐쾾쐞븯寃 궗슜릺怨 엳떎. 湲곗〈뿉 궗슜릺뿀뜕 쑀쟾옄 蹂삎 諛⑸쾿 留덉슦뒪 諛곗븘 以꾧린(ES) 꽭룷뿉꽌 긽룞 옱議고빀쓣 씠슜븳 諛⑸쾿쑝濡 鍮꾩슜씠 留롮씠 뱾怨 삤옖 떆媛꾧낵 留롮 끂룞씠 븘슂뻽떎. 븯吏留 Zinc-finger nucleases (ZFNs) transcription activator-like effector nucleases (TALENs) 媛숈 site-specific nuclease, 利 씤怨 쑀쟾옄 媛쐞媛 媛쒕컻맖쑝濡쒖뜥 듅젙 쑀쟾옄 쐞移섏뿉꽌 DNA쓽 씠以 媛떏쓣 옄瑜닿퀬 렪吏묓븷 닔 엳寃 릺뿀쑝硫 씠뒗 쑀쟾怨듯븰뿉 以묒슂븳 諛쒖쟾쓣 씠걣寃 릺뿀떎(Bibikova et al., 2003; Carlson et al., 2012; Cho et al., 2013). 쑀쟾옄 媛쐞 以 媛옣 理쒓렐뿉 諛앺吏 clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated protein 9 (Cas9) system 꽭洹좉낵 怨좎꽭洹 궡뿉 議댁옱븯뒗 쟻쓳 硫댁뿭 떆뒪뀥뿉꽌 쑀옒븳 寃껋쑝濡 RNA瑜 湲곕컲쑝濡 옉슜븳떎(Doudna and Charpentier, 2014). Plasmid DNA굹 virus 媛숈 쇅遺 DNA媛 꽭洹좎뿉 移⑥엯뻽쓣 븣 씠뱾 DNA 꽌뿴쓽 씪遺瑜 湲곗뼲븯怨 諛섎났 媛먯뿼 떆 CRISPR RNAs (crRNA) transactivating crRNA (tracrRNA)媛 삎꽦맂떎. 빀꽦맂 crRNA, tracrRNA Cas9 떒諛깆쭏 蹂듯빀泥닿 삎꽦릺硫 쇅遺 DNA 꽌뿴쓣 듅씠쟻쑝濡 젅떒븿쑝濡쒖뜥 쇅옒 DNA瑜 젣嫄고븳떎(Hsu et al., 2013; Ran et al., 2013). Cas9 nuclease뒗 Streptococcus pyogenes뿉꽌 쑀옒븳 떒諛깆쭏濡 crRNA쓽 spacer뿉 빐떦븯뒗 20 bp 뿼湲곗꽌뿴 諛붾줈 뮘뿉 議댁옱븯뒗 protospacer adjacent motif (PAM) 뿼湲곗꽌뿴 5'-NGG-3'쓣 씤떇븯뿬 PAM 꽌뿴 諛붾줈 븵쓣 젅떒븯뿬 쇅옒 DNA瑜 젣嫄고븳떎. 뵲씪꽌 CRISPR/Cas9 system쓣 씠슜븳 쑀쟾옄 렪吏묒쓣 쐞빐꽌뒗 Cas9 떒諛깆쭏怨 렪吏묓븯怨좎옄 븯뒗 遺쐞쓽 crRNA tracrRNA媛 븘슂븯硫, crRNA tracrRNA쓽 븘닔쟻씤 遺遺꾩쓣 뿰寃고븯뿬 븯굹쓽 single guide RNA (sgRNA) 삎깭濡 젣怨듯븷 닔 엳떎(Horii et al., 2014). 씠 媛숈씠 CRISPR/Cas9 system 옉 뿼湲곗뙇 媛씠뱶 RNA (sgRNA) Cag9 떒諛깆쭏쓣 궗슜븯뿬 쇅옒 DNA瑜 遺쐞-듅씠쟻씤 諛⑹떇쑝濡 몴쟻솕븯怨 젅떒븯뒗 諛⑸쾿쑝濡 돺怨 鍮좊Ⅴ湲 븣臾몄뿉 留롮 뿰援ъ옄뱾씠 씠 湲곗닠뿉 二쇰ぉ븯怨 씠슜븯怨 엳떎. 뜑슧씠 븳 媛쒖쓽 뿼湲곗꽌뿴쓣 移섑솚븯뒗 諛⑸쾿怨 븳 踰덉뿉 뿬윭 쑀쟾옄뿉 룎뿰蹂씠媛 깮꽦맂 깮伊먯쓽 젣옉룄 媛뒫븯寃 릺뿀떎(Wang et al., 2013; Aida., 2016; Ma et al., 2017).

빆泥대뒗 醫낆쥌 硫댁뿭湲濡쒕텋由곗쑝濡 遺덈━硫 빆썝怨 寃고빀븯뿬 빆썝쓽 옉슜쓣 諛⑺빐븯嫄곕굹, 빆썝쓣 젣嫄고븯뒗 硫댁뿭 떒諛깆쭏씠떎. 빆泥댁뿉뒗 immunoglobulin(Ig)G, IgM, IgA, IgE 諛 IgD쓽 5醫낅쪟媛 엳떎. 씠뱾 紐⑤몢뒗 湲곕낯쟻쑝濡 4媛쒖쓽 궗뒳濡 맂 빆泥 援ъ“瑜 媛뽰留, 媛곴컖 以묒뇙 遺덈쁺뿭 쑀쟾옄 μ, δ, γ, α, ε濡쒕꽣 留뚮뱾뼱吏 以묒뇙瑜 룷븿븳떎(Lonberg and Huszar, 1995; Beck et al., 2010). 궗엺쓽 삁泥 궡 二쇰맂 빆泥대뒗 IgG씠硫 援ъ“ 湲곕뒫硫댁뿉꽌 媛옣 옒 븣젮졇 엳怨, IgG뒗 떎떆 IgG1, IgG2, IgG3, IgG4 isotype濡 굹돇硫 媛곴컖쓽 援ъ“ 諛 湲곕뒫쟻 듅꽦 떎瑜대떎. IgG뒗 以묒뇙(heavy chain, 50 kDa) 떒諛깆쭏 2媛쒖 寃쎌뇙(light chain, 25 kDa) 떒諛깆쭏 2媛쒕줈 留뚮뱾뼱吏 Y옄 紐⑥뼇쓽 留ㅼ슦 븞젙맂 援ъ“(遺꾩옄웾, 150 kDa)쓣 삎꽦븯怨 엳떎. 궗엺怨 깮伊먯쓽 빆泥 쑀쟾옄뒗 援ъ“쟻, 湲곕뒫쟻쑝濡 留ㅼ슦 쑀궗븿씠 諛앺졇 엳떎(Lonberg and Huszar, 1995).

蹂 뿰援ъ뿉꽌뒗 CRISPR-Cas9 system쓣 씠슜븯뿬 씤媛꾧낵 湲곕뒫쟻쑝濡 쑀궗븳 빆泥대줈 븣젮吏 mouse IgG2a 옄由ъ뿉 human IgG1쓽 light kappa constant 遺쐞瑜 궫엯떆궓 留덉슦뒪(hIgG Lc KI mice)瑜 젣옉븯怨좎옄 븯떎. CRIRPR-Cas9 system 留롮 뿰援ъ옄뱾씠 뿰援ъ뿉 씠슜븯怨 엳쑝굹 븘吏 媛쒕컻 珥덇린 떒怨꾨줈 留롮 떆뻾 李⑹삤 솗由쎈맂 protocol씠 議댁옱븯怨 엳吏 븡떎. 蹂 뿰援ъ뿉꽌뒗 CRISPR/Cas9 system쓣 씠슜븳 쑀쟾옄 궫엯(knock-in) 룞臾 깮궛뿉 슚怨쇱쟻씤 諛⑸쾿쓣 젣떆븯怨, 뼢썑 씤媛 빆泥대 깮궛븷 닔 엳뒗 留덉슦뒪 깮궛쓣 쐞븳 湲곗큹瑜 留덈젴븯怨좎옄 븯떎.

옱猷 諛 諛⑸쾿

Mouse human IgG1 hybrid light kappa constant chain DNA 젣옉

誘멸뎅援由쎌깮臾쇱젙蹂댁꽱꽣(NCBI)뿉꽌 human IgG1 빆泥 쑀쟾옄쓽 light kappa chain 뿼湲곗꽌뿴쓣 솗씤(NC_000002.12)븯쑝硫, 留덉슦뒪 IgG2a light kappa chain 꽌뿴(NC_000072.6)쓣 鍮꾧탳 遺꾩꽍븯떎. 빟 320 bp쓽 kappa constant 꽌뿴쓣 human 꽌뿴濡 泥댄븯怨 homologous recombination (HR)씠 씪뼱궇 닔 엳룄濡 5' HR arm怨 3' HR arm쓣 媛 1.25 kb 젙룄 룷븿븳 留덉슦뒪 뿼湲곗꽌뿴 (2,803 bp)쓣 pUC57 vector쓽 EcoRI 젣븳슚냼 遺쐞瑜 씠슜븯뿬 궫엯릺룄濡 二쇰Ц 젣옉븯떎(Bioneer, Korea).

In vitro RNA transcription

sgRNA쓽 design (Table 1)怨 in vitro transcription zhang's laboratory뿉꽌 젣怨듯븯뒗 諛⑸쾿쑝濡 떎떆븯떎(http://www. genome-engineering.org/crispr). 媛곴컖쓽 guide RNA oligomer瑜 annealing 븳 썑 pUC57-sg RNA expression vector (Life Technologies, USA)쓽 Bsa I 젣븳슚냼 遺쐞뿉 궫엯븯떎. Guide RNA 꽌뿴씠 삎吏덉쟾솚맂 plasmid瑜 sequencing쓣 넻빐 솗씤븯떎. 솗씤맂 clone sgRNA쓽 in vitro transcription template濡 MEGAshortscript T7 kit (Life Technologies, USA)쓣 씠슜븯뿬 transcription 떆궓 썑 RNA뒗 MEGAclear kit (Life Technologies, Carlsbad, CA, USA)怨 elution buffer瑜 씠슜빐 遺꾨━뻽떎. 遺꾨━맂 sgRNA뒗 NanoDrop (Thermo Scientific, USA)쓣 씠슜빐 냽룄 quality瑜 痢≪젙븯떎.

Sequence of crRNA, tracrRNA, sgRNA oligomer used in this study

OligoSequence
Kappa F1 crRNA5'-GGUGGGAAGAUGGAUACAGUGUUUUAGAGCUAUGCUGUUUUG-3'
Kappa B1 crRNA5'-CCAUUGUCAAGAGCUUCAACGUUUUAGAGCUAUGCUGUUUUG-3'
tracrRNA5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3'
Kappa F15'-TAGGGGTGGGAAGATGGATACAGT-3'
Kappa B15'-TAGGGCCATTGTCAAGAGCTTCAAC-3'

Microinjection

C57BL/6 wt怨 ICR mouse(삤由ъ뿏듃 諛붿씠삤, 븳援)瑜 궗슜븯쑝硫, 紐⑤뱺 룞臾 떎뿕 怨좊젮븰援 깮紐낃났븰썝 怨꾨┝떎뿕룞臾 옄썝꽱꽣뿉꽌 吏꾪뻾븯떎. 삉븳 怨좊젮븰援 떎뿕룞臾 쑄由ъ쐞썝쉶쓽 듅씤쓣 諛쏆븘 듅씤맂 吏移⑥뿉 뵲씪 쑄由ъ쟻 湲곗뿉 쓽嫄고븯뿬 떆뻾븯떎(KUIACUC-2018-25). hIgG Lc KI 留덉슦뒪 젣옉쓣 쐞븳 microinjection C57BL/6 4二쇰졊 븫而 留덉슦뒪瑜 씠슜븯쑝硫 7.5 unit PMSG (Prospec, Israel)瑜 蹂듦컯 二쇱궗 썑 48떆媛 뮘뿉 룞웾쓽 hCG (Prospec, Israel) 샇瑜대が쓣 蹂듦컯 二쇱궗븯뿬 怨쇰같쓣 쑀룄븯떎. 怨쇰같씠 쑀룄맂 留덉슦뒪뒗 C57BL/6 닔而 留덉슦뒪 븯猷삳룞븞 援먮같 썑 떎쓬 궇 vaginal plug쓣 泥댄겕븯뿬 援먮같 쑀臾대 솗씤븯뿬 Plug씠 솗씤맂 븫而 留덉슦뒪쓽 궃愿뿉꽌 닔젙쓣 遺꾨━뻽떎(Ittner and Gotz, 2007). 닔젙쓽 쟾빑뿉 以鍮꾨맂 target donor DNA, sgRNA, 삉뒗 crRNA, tracrRNA, Cas9 protein mixture瑜 microinjection쑝濡 二쇱엯븯떎. 37℃ 諛곗뼇湲곗뿉꽌 닔젙씠 2 cell濡 遺꾪솕릺뒗 寃껋쓣 쁽誘멸꼍쑝濡 솗씤븳 썑, 媛엫떊 맂 ICR 븫而 留덉슦뒪쓽 궃愿 뙺遺뿉 10~ 20媛쒖쓽 닔젙쓣 씠떇븯떎. 3二 썑 깭뼱굹뒗 깉겮 留덉슦뒪쓽 닽옄瑜 솗씤븯怨 4二 썑뿉 瑗щ━쓽 씪遺瑜 옒씪 쑀쟾옄瑜 遺꾩꽍쓣 떎떆븯떎.

쑀쟾옄 궫엯 솗씤쓣 쐞븳 primer 꽑諛 諛 PCR 솗씤

Human IgG1 light kappa constant 쑀쟾옄媛 궫엯맂 留덉슦뒪 솗씤쓣 쐞빐 primer瑜 젣옉븯쑝硫(Fig. 2, Table 2), 쑀쟾옄 궫엯 PCR 쑀쟾옄 꽌뿴 遺꾩꽍쓣 넻빐 理쒖쥌 솗씤븯떎. 쑀쟾옄 궫엯 쑀臾 솗씤 microinjection뿉 쓽빐 깭뼱궃 깉겮뱾씠 4二쇰졊씠 릺뿀쓣 븣 떎떆븯쑝硫 瑗щ━쓽 씪遺瑜 옒씪 genomic DNA瑜 異붿텧븯뿬 PCR쓣 넻빐 솗씤뻽떎. PCR 議곗꽦 AccuPower® HotStart PCR PreMix (Bioneer, Korea), 媛 primer (25 pmol) 媛 1 μL, genomic DNA 1 μL 諛 DW 17 μL瑜 mix븯뿬 理쒖쥌 20 μL濡 떎떆뻽떎, 諛섏쓳 議곌굔 95℃ 5遺, 蹂꽦 썑 95℃뿉꽌 30珥, 57.5℃ 30珥, 72℃ 70珥덈 珥 30쉶 諛섎났 닔뻾 썑 72℃ 5遺꾩쑝濡 떎떆븯쑝硫, PCR 궛臾쇱 1% agarose gel뿉꽌 100 v 30遺 룞븞 쟾湲곗쁺룞 썑 寃곌낵瑜 솗씤븯떎. 1李 PCR怨 2李 PCR쓣 솗씤 썑 PCR 궛臾쇱 sequencing (Bioneer, Korea)쓣 넻빐 理쒖쥌 뿼湲곗꽌뿴쓣 솗씤븯뿬 留덉슦뒪 IgG2a light kappa constant 꽌뿴씠 human light kappa constant 꽌뿴濡 泥대맂 寃껋쓣 솗씤븯떎.

Primers used in this study

Primer nameSequence
mKLC-F15'-ACC AGG GTC TGA TGA ATT GC-3'
mKLC-F25'-GTG CAT CCT GGC CCC ATT GTT CC-3'
mKLC-R15'-CC AGA TGT TAA CTG CTC ACT G-3'
mKLC-R25'-TGT ACT TTG AGC TCT GGA AGG C-3'
hKLC-F15'-G TGG AAG GTG GAT AAC GCC CT-3'
hKLC-R15'-T ACC CGA TTG GAG GGC GTT-3'

Fig. 2. Design and construction for human IgG1 light kappa Knock-In mouse.

(A) In order to replace the mouse IgG1 light kappa constant gene (mIgG Lc) with the human IgG1 light kappa constant gene (hIgG Lc), Kappa F1 and Kappa B1 crRNAs were selected to recognize and cut of Cas9 protein and homologous recombination was used to increase insertion efficiency with hIgG Lc. The outer genomic DNA sequence of mIgG Lc was inserted into the 5 'arm and the 3' arm by about 1.2 Kb to design a recombinant allele. (B) The designed Recombinant allele gene was used to synthesize hIgG1 Lc KI vector as donor DNA for Knock-In mouse construction.


쑀쟾옄 諛쒗쁽 솗씤

Human light kappa constant 쑀쟾옄媛 궫엯맂 留덉슦뒪뒗 닔而룹쓽 寃쎌슦 6二쇰졊 씠썑 C57BL/6 wt female怨 援먮같瑜 떎떆븯쑝硫, 븫而룹 8二쇰졊 씠썑 닔而룰낵 留덉갔媛吏濡 C57BL/6 wt male怨 援먮같瑜 떎떆븯떎. 뿬윭 李⑤ 援먮같瑜 넻빐 뼸뼱吏 留덉슦뒪濡쒕꽣 human IgG light kappa 쑀쟾옄 諛쒗쁽쓣 솗씤븯湲 쐞빐 瑗щ━濡쒕꽣 PBMC瑜 梨꾩랬븯뿬 FACS 뿼깋쓣 떎떆븯떎. FACS 뿼깋쓣 쐞빐 ammonium chloride-potassium (ACK) 슜븸怨 븿猿 10遺꾧컙 떎삩뿉꽌 諛곗뼇븳 뮘, phosphate-buffered saline (PBS)濡 꽭젙븳 떎쓬, Fc receptor瑜 諛쒗쁽븯뒗 꽭룷뱾뿉 쓽븳 鍮꾪듅씠쟻 諛섏쓳쓣 以꾩씠湲 쐞빐 anit-FcrRII/III mAb (2.4G2)瑜 씠슜븯뿬 4℃뿉꽌 15遺꾧컙 諛섏쓳떆耳곕떎. 쑀꽭룷 遺꾩꽍쓣 쐞빐 FITC-conjugated anti-mouse B220 (BD Biosciences, USA), PE-conjugated anti-mouse Igk (BD Biosciences, USA), APC-conjugated anti-human Igk B220 (BD Biosciences, USA) 빆泥대 媛곴컖 泥섎━븯뿬 4℃뿉꽌 30遺꾧컙 諛섏쓳븯떎. 빆泥 諛섏쓳씠 걹궃 꽭룷瑜 PBS濡 꽭泥숉븯怨, 1% paraformaldehyde 300 μL뿉 怨좎젙븯뿬, 쑀꽭룷 遺꾩꽍湲 (FACSCalibur; USA)濡 遺꾩꽍븯떎. 遺꾩꽍 봽濡쒓렇옩쑝濡쒕뒗 CellQuest sof tware (BD Biosciences)瑜 궗슜븯떎.

寃 怨

Humanized IgG1 light kappa constant region Knock-In 깮伊 젣옉쓣 쐞븳 뵒옄씤

궗엺쓽 빆泥대 깮궛븯뒗 留덉슦뒪 젣옉쓣 쐞빐 蹂 뿰援ъ뿉꽌뒗 留덉슦뒪 IgG light kappa constant 遺쐞瑜 癒쇱 씤媛꾩쓽 쑀쟾옄濡 泥댄븯怨좎옄 븯떎. CRISPR/Cas9 system쓣 씠슜븯뿬 留덉슦뒪 IgG1 kappa constant region쓣 human IgG kappa constant region쑝濡 泥 궫엯맂 留덉슦뒪瑜 젣옉븯뿬 뼢썑 씤媛꾩쓽 빆泥대 깮궛븯怨좎옄 븯떎. 留덉슦뒪 IgG1 light kappa constant 쑀쟾옄뒗 6踰 뿼깋泥댁뿉 쐞移섑븯硫 320 bp (Gene ID: 16071)濡 援ъ꽦릺뼱 엳쑝硫, human IgG light kappa constant region 2踰 뿼깋泥댁뿉 쐞移섑븯硫 323 bp (Gene ID: 3514)쓽 겕湲곕 媛吏怨 엳떎. 몢 쑀쟾옄뒗 빟 71% 젙룄쓽 긽룞꽦쓣 媛吏怨 엳쑝硫, 삁븸 궡 媛옣 뭾遺븯硫 留ㅼ슦 쑀궗븳 湲곕뒫쓣 븳떎. 쑀쟾옄 궫엯쓽 슚쑉쓣 利앷떆궎湲 쐞빐 mouse IgG1 light kappa 쑀쟾옄 遺쐞뿉 hIgG light kappa 쑀쟾옄 꽌뿴쓣 궫엯븯怨, Fig. 1A쓽 recombinant allele怨 媛숈씠 mouse IgG1 light kappa region 諛붽묑履쎌뿉 쐞移섑븳 꽌뿴쓽 5' HR arm怨 3' HR arm쓣 빟 1.2 kb뵫 mouse 꽌뿴쓣 궫엯븯뿬 긽룞옱議고빀씠 씪뼱 궇 닔 엳룄濡 꽕怨꾪븯떎(Fig. 1A). 꽕怨꾨맂 쑀쟾옄뒗 humanized IgG1 light kappa chain쓣 諛쒗쁽븯뒗 留덉슦뒪瑜 젣옉븯湲 쐞븳 donor vector濡 씠슜븯怨좎옄 DNA (hIgG Lc KI vector)瑜 빀꽦븯떎(Fig. 1B). CRISPR/Cas9 system쓣 씠슜븳 쑀쟾옄 렪吏 湲곕뒫 Cas9 떒諛깆쭏씠 옄瑜 닔 엳뒗 PAM 꽌뿴쓣 媛吏 target guide RNA 꽑젙씠 留ㅼ슦 以묒슂븯떎. 3' 留먮떒뿉 NGG瑜 젣쇅븳 17~24 bp뒗 40~80%쓽 GC 븿웾쓣 룷븿븯硫 삉븳 湲몄씠媛 吏㏃쓣닔濡 off target 슚怨쇨 理쒖냼솕릺뒗 寃껋쑝濡 븣젮졇 엳떎. Guide RNA 꽌뿴 RGEN怨 MIT 媛숈 쎒뿉꽌 젣怨듯븯뒗 꽌뿴쓣 李멸퀬 · 鍮꾧탳븯뿬 꽑젙븯쑝硫(Table 1), sgRNA瑜 쐞븳 oligomer뒗 諛붿씠삤땲븘뿉꽌 빀꽦븯怨, crRNA tracrRNA뒗 떎留덉퐯뿉꽌 빀꽦븯떎. Cas9 떒諛깆쭏씠 씤떇빐꽌 옄瑜대뒗 target 遺쐞뒗 human light kappa chain쓽 5' 留먮떒(15踰덉㎏)怨 3' 留먮떒(308踰덉㎏)뿉 쐞移섑븯뿬 human 쑀쟾옄 넀떎쓣 理쒖냼솕븯떎(Fig. 1A).

Fig. 1.

Map of specific PCR primers for confirm of hIgG Lc KI mouse.


hIgG1 Lc Knock-In 留덉슦뒪 젣옉쓣 쐞븳 microinjection

理쒓렐 CRISPR/Cas9 system쓣 씠슜븳 gene editing 留롮 뿰援ъ옄뱾쓣 넻빐 솢諛쒗븯寃 뿰援ш 吏꾪뻾릺怨 엳떎. 븯吏留 삎吏덉쟾솚 룞臾 깮궛 遺꾩빞뿉꽌뒗 鍮꾩슜怨 떆꽕쟻씤 痢〓㈃뿉꽌 젒洹쇱씠 돺吏 븡湲 븣臾몄뿉 留ㅼ슦 븳젙쟻씤 뿰援ш 吏꾪뻾릺怨 엳떎. 뵲씪꽌 CRISPR/Cas9 system쓣 씠슜븳 슚쑉쟻씤 삎吏덉쟾솚 룞臾 젣옉 諛⑸쾿쓽 젣떆뒗 留ㅼ슦 以묒슂븯떎. 씠踰 뿰援ъ뿉꽌뒗 CRISPR/Cas9 system쓣 씠슜븯뿬 썝븯뒗 쑀쟾옄瑜 궫엯븯뒗 Knock-In 留덉슦뒪瑜 젣옉븷 븣 슚쑉쓣 利앷떆궎뒗 諛⑸쾿쓣 李얘퀬옄 븯떎. Cas9 떒諛깆쭏(NEB, USA)怨 donor DNA (hIgG Lc KI vector)瑜 湲곕낯쑝濡 씠슜븯쑝硫, target guide RNA瑜 sgRNA crRNA/tracrRNA 삎깭濡 씠슜뻽쓣 븣 깮궛릺뒗 KI 留덉슦뒪 슚쑉쓣 鍮꾧탳븯떎. 삉븳 microinjection 떆뿉 donor DNA쓽 냽룄 Cas9 떒諛깆쭏쓽 냽룄瑜 100 ng/μL, 10 ng/μL쓽 議고빀쑝濡 떎떆븯뿬 鍮꾧탳븯떎. Donor DNA瑜 100 ng/μL濡 怨좊냽룄瑜 궗슜뻽쓣 寃쎌슦, egg transfer 썑뿉 pup씠 깮꽦릺吏 븡븘 鍮꾧탳뿉꽌 젣쇅븯떎(data not shown). KI 留덉슦뒪 깮궛 슚쑉 鍮꾧탳瑜 쐞빐 癒쇱 Kappa F1 sgRNA 2.5 ng/μL, Kappa B1 sgRNA 2.5 ng/μL Cas9 떒諛깆쭏 100 ng/μL 삉뒗 10 ng/μL, 10 ng/μL donor DNA瑜 닔젙쓽 쟾빑뿉 microinjection쑝濡 二쇱엯븯떎. 떎쓬쑝濡쒕뒗 Kappa F1 crRNA 0.6 pmol/μL, Kappa B1 crRNA 0.6 pmol/μL, tracrRNA 0.6 pmol/μL Cas9 떒諛깆쭏 100 ng/μL 삉뒗 10 ng/μL, 10 ng/μL donor DNA瑜 닔젙쓽 쟾빑뿉 microinjection쓣 떎떆뻽떎. sgRNA瑜 二쇱엯븳 썑 쟾泥 104留덈━쓽 깉겮媛 깭뼱궗쑝硫, crRNA tracrRNA쓣 二쇱엯븳 寃쎌슦뿉뒗 쟾泥 132留덈━쓽 깉겮瑜 뼸쓣 닔 엳뿀떎(Table 3). Target guide RNA쓽 李⑥씠씤 sgRNA굹 crRNA/tracrRNA瑜 궗슜뻽쓣 븣, 닔젙쓣 由щえ뿉 씠떇 썑 깉겮瑜 뼸쓣 븣源뚯뒗(Transfer 슚쑉: 87.8% vs. 92.5%, P=0.08, newborn 슚쑉: 9.4% vs. 10.5%, P=0.06) 겙 李⑥씠瑜 蹂댁씠吏 븡븯떎(Table 3).

Summary of CRISPR/Cas9 mediated Knock-In mouse

Guide RNACas9 protein concInjected eggTransferred (%)Newborn (%)1st PCR targeted (%)2nd PCR Knock-In (%)
sgRNA100 ng/μL681608 (89.3)54 (8.9)3 (5.5)0 (0)
sgRNA 10 ng/μL594512 (86.2)50 (9.8)4 (8)0 (0)
crRNA and tracrRNA100 ng/μL613554 (90.4) 58 (10.5)21 (36.2) 1 (1.7)
crRNA and tracrRNA 10 ng/μL718679 (94.6) 74 (10.9)27 (36.5) 3 (4.1)

hIgG1 Lc Knock-In 留덉슦뒪 솗씤

sgRNA 二쇱엯怨 crRNA/tracrRNA 二쇱엯쓣 넻빐 뼸뼱吏 깉겮뱾濡쒕꽣 human IgG1 light kappa constant 쑀쟾옄쓽 궫엯쓣 솗씤븯湲 쐞빐 4二쇰졊씠 맆 븣源뚯 湲곕떎由 썑 瑗щ━쓽 씪遺瑜 옒씪 genomic DNA瑜 異붿텧븯떎. 癒쇱 1李 PCR濡 mKLC-F1怨 hKLC-R1 primer瑜 씠슜븯뿬 human IgG light kappa chain쓽 궫엯쓣 솗씤븯떎(690 bp PCR 궛臾). 1李 PCR 寃곌낵 sgRNA瑜 二쇱엯븳 썑 뼸뼱吏 104留덈━쓽 깉겮 以 Cas9 떒諛깆쭏쓣 100 ng/μL瑜 궗슜뻽쓣 븣 3留덈━, Cas9 떒諛깆쭏쓣 10 ng/μL瑜 궗슜뻽쓣 븣 4留덈━뿉꽌 쑀쟾옄 궫엯 씠 솗씤릺뿀떎. 삉븳 crRNA/tracrRNA 100 ng/μL쓽 Cas9 떒諛깆쭏쓣 二쇱엯븳 썑 뼸뼱吏 58留덈━ 깉겮 以 21留덈━媛 1李 PCR뿉꽌 쑀쟾옄 궫엯씠 솗씤릺뿀쑝硫, 10 ng/μL Cas9 떒諛깆쭏怨 crRNA 議고빀쓣 궗슜뻽쓣 븣뒗 쟾泥 74留덈━ 以 27留덈━媛 1李 PCR뿉꽌 쑀쟾옄 궫엯씠 솗씤릺뿀떎(Fig. 3A). 1李 PCR뿉꽌 솗씤맂 54留덈━ 以 젙솗븳 쐞移섏뿉 궫엯릺뿀뒗吏 솗씤쓣 쐞빐 2李 PCR쓣 mKLC-F2怨 mKLC-R1 primer瑜 씠슜븯뿬 솗씤븯떎(Fig. 3A). 궫엯맂 꽌뿴뿉 mutation씠 뾾뒗吏 뿼湲곗꽌뿴 솗씤쓣 쐞빐 mKLC-F2怨 hKLC-R1 primer hKLC-F1怨 mKLC-R2 primer瑜 씠슜븯뿬 PCR쓣떎떆븳 썑 뿼湲곗꽌뿴 遺꾩꽍쓣 떎떆븯떎. 2李 PCR 寃곌낵 sgRNA瑜 二쇱엯븳 썑 뼸 7留덈━뿉꽌뒗 썝븯뒗 쐞移섏뿉 쑀쟾옄 궫엯쓣 솗씤븷 닔 뾾뿀쑝硫, crRNA/tracrRNA 100 ng/μL Cas9 떒諛깆쭏 二쇱엯쓣 넻빐 뼸 21留덈━ 以 1留덈━ crRNA/tracrRNA 10 ng/μL Cas9 떒諛깆쭏 二쇱엯쓣 넻빐 뼸 27由 以 3留덈━뿉꽌 쑀쟾옄 궫엯쓣 솗씤(1.6 kb)븯쑝硫, 뿼湲곗꽌뿴 遺꾩꽍쓣 넻빐 젙긽쟻씤 쑀쟾옄엫쓣 솗씤븯떎(Fig. 3B).

Fig. 3. Analysis of hIgG Lc KI mice.

(A) Mice born through the CRISPR / Cas9 system cut off a portion of the tail at 4 weeks of age and confirmed the gene KI by PCR. In the first PCR, human gene insertion (690 bp) was confirmed using mKLC-F1 and hKLC-R1 primers. In the first PCR, the mice inserted into the gene were confirmed to be inserted (1.6 Kb) at the desired position by the second PCR (mKLC-F2 and mKLC-R1 primers). (B) Mouse confirmed by the secondary PCR confirmed that the normal gene (humanized IgG KI) is inserted through sequencing.


Human IgG light kappa chain 쑀쟾옄 諛쒗쁽 솗씤

Fig. 3뿉꽌 媛숈씠 1 · 2李 PCR怨 뿼湲곗꽌뿴 遺꾩꽍쓣 넻빐 human IgG light kappa constant 쑀쟾옄媛 궫엯맂 寃껋쓣 솗씤븯떎. 떎쓬쑝濡쒕뒗 human IgG light kappa constant 쑀쟾옄 諛쒗쁽쓣 솗씤븯떎. 쑀쟾옄쓽 궫엯씠 씪뼱굹뜑씪룄 諛쒗쁽씠 릺吏 븡뒗 寃쎌슦뿉뒗 빆泥대 깮궛븯뒗 留덉슦뒪濡쒖꽌 湲곕뒫쓣 븷 닔 뾾湲 븣臾몄뿉 궫엯맂 쑀쟾옄媛 留덉슦뒪 깮泥 궡뿉꽌 諛쒗쁽릺뒗吏瑜 솗씤븯뒗 寃껋씠 以묒슂븯떎. 씠瑜 쐞빐 hIgG Lc KI 留덉슦뒪뒗 뿬윭 李⑤ 援먮같媛 吏꾪뻾릺뿀쑝硫, 援먮같 썑 뼸뼱吏 깉겮쓽 PBMC瑜 遺꾨━븯뿬 B 꽭룷뿉꽌 human IgG kappa chain쓽 諛쒗쁽쓣 FACS 뿼깋쓣 넻빐 솗씤븯떎. Fig. 4 媛숈씠 hIgG Lc+/-(ID#7321)뒗 33.6%쓽 mouse Ig kappa chain쓣 諛쒗쁽븯硫 40.9%쓽 human Ig kappa chain쓣 諛쒗쁽븯뒗 諛섎㈃, hIgG Lc+/+ (ID#8611)뒗 85.5%쓽 human Ig kappa chain留뚯쓣 諛쒗쁽뻽떎. 寃곌낵쟻쑝濡 CRISPR/Cas9 system쓣 넻빐 젣옉맂 hIgG Lc Knock-In 留덉슦뒪媛 젙긽쟻쑝濡 씤媛 빆泥 떒諛깆쭏쓣 諛쒗쁽븯뒗 寃껋쓣 솗씤븷 닔 엳뿀떎.

Fig. 4. Human IgG light kappa chain is expressed in B cells of hIgG Lc KI mice.

Human IgG light kappa constant gene expression was confirmed from KI mice inserted with hIgG Lc gene. PBMCs of wild type mice and hIgG Lc KI mice were stained with mB220, mIgK and hIgk antibodies, and the expression of mouse IgG kappa chain and human IgG kappa chain among B cells was confirmed through FACS analysis.


怨 李

移섎즺슜 빆泥대뒗 1994뀈 泥 빆泥 移섎즺젣媛 媛쒕컻 · 긽뭹솕 맂 씠옒濡 媛옣 鍮좊Ⅸ 꽦옣쓣 蹂댁씠怨 엳뒗 쓽빟뭹 以 븯굹濡 떎뼇븳 옄媛硫댁뿭吏덊솚, 븫, 湲됱꽦 怨⑥닔 諛깊삁蹂, 嫄댁꽑, 怨⑤떎怨듭쬆 移섎즺뿉 씠슜릺怨 엳떎. 珥덇린 移섎즺젣뒗 떒諛깆쭏怨듯븰 諛쒖쟾뿉 쓽븳 옱議고빀 떒諛깆쭏 빆泥 移섎즺젣 삎깭쑝굹, 理쒓렐뿉뒗 씤媛 빆泥 쑀쟾옄瑜 씠떇븳 삎吏덉쟾솚 留덉슦뒪濡쒕꽣 빆썝 듅씠쟻씤 씤媛 떒씪겢濡 빆泥대 젣議고븯뒗 湲곗닠源뚯 諛쒖쟾븯떎(Deng and Capecchi, 1992; Green et al., 1994; Lonberg, 2008). 誘멸뎅씠굹 씪蹂몄뿉꽌뒗 씤媛 빆泥대 깮궛븷 닔 엳뒗 삎吏덉쟾솚 留덉슦뒪瑜 씠슜븳 移섎즺슜 빆泥닿 쁽옱 떆뙋 以묒씠嫄곕굹 엫긽 떆뿕떒怨꾩뿉 엳뒗 빆泥닿 50뿬醫낆뿉 씠瑜닿퀬 엳쓣 留뚰겮 솢諛쒗븳 뿰援ш 吏꾪뻾 以묒씠떎(Green, 1999; Lonberg, 2008). 븯吏留 援궡뿉꽌뒗 삎吏덉쟾솚 留덉슦뒪 깮궛湲곗닠젰쓽 븳怨꾩 怨좉쓽 鍮꾩슜, 젣옉뿉 냼슂릺뒗 湲곌컙, 떆꽕 쑀吏 벑쓽 臾몄젣濡 씤媛꾩쓽 빆泥대 깮궛븯뒗 留덉슦뒪뿉 븳 뿰援ш 吏꾪뻾릺吏 븡怨 엳떎. 궗엺쓽 빆泥대 깮궛븯뒗 留덉슦뒪媛 젣옉릺硫 湲곗〈뿉 떆뙋릺吏 븡 떎뼇븳 빆썝뿉 뵲瑜 빆泥대 깮궛븷 닔 엳쑝硫, 援쇅뿉꽌 蹂댁쑀븯怨 엳뒗 humanized Ab 留덉슦뒪 젣옉 湲곕쾿怨 빆泥 쑀쟾옄 뵒옄씤쓽 李⑥씠濡 李⑤퀎꽦쓣 媛吏湲 븣臾몄뿉 蹂 뿰援ъ뿉꽌 젣옉븯怨좎옄 븯뒗 human Ig KI 留덉슦뒪룄 異⑸텇븳 寃쎌웳젰쓣 엳쓣 寃껋쑝濡 湲곕맂떎.

Cas9 떒諛깆쭏怨 sgRNA瑜 궗슜븯硫 쟾궗 踰덉뿭怨쇱젙씠 깮왂릺湲 븣臾몄뿉 鍮좊Ⅸ 떆媛 궡뿉 룎뿰蹂씠媛 쑀룄릺怨 떒 떆媛 룞븞뿉留 쑀쟾泥닿 CRISPR/Cas9뿉 끂異쒕릺뼱 off-target씠 以꾩뼱뱶뒗 寃껋쑝濡 븣젮졇 엳떎(Fu et al., 2013; Shen et al., 2014). CRISPR/Cas9 system쓣 씠슜븳 떎뼇븳 쑀쟾옄 렪吏묒씠 씤媛꾨같븘以꾧린 꽭룷瑜 鍮꾨’븯뿬 留덉슦뒪, drosophila, zebrafish, rice, Arabidopsis 벑 룞 · 떇臾쇱쓽 紐⑤뜽 떆뒪뀥뿉꽌 솢諛쒗븳 뿰援щ뱾씠 吏꾪뻾릺怨 엳떎(Shan et al., 2013; Fu et al., 2013; Yan et al., 2014; Chu et al., 2015). 듅엳 삎吏덉쟾솚 留덉슦뒪 젣옉뿉꽌 떎뼇븳 삎깭쓽 쑀쟾옄 렪吏묒씠 씠猷⑥뼱吏硫댁꽌 CRISPR/Cas9 system쓽 쟻슜 諛⑸쾿뿉 뵲瑜 젙솗꽦怨 쟻以 슚쑉뿉 빐 젣떆븯怨 엳떎(Horii et al., 2014; Chu et al., 2016; Raveux et al., 2017). 쑀쟾옄瑜 젣嫄고븯뒗 諛⑸쾿 鍮꾧탳쟻 넂 슚쑉쓽 쑀쟾옄 렪吏묒씠 媛뒫븯硫 몴以솕맂 諛⑸쾿룄 媛뒫븯떎. 븯吏留 썝븯뒗 遺쐞뿉 썝븯뒗 쑀쟾옄瑜 궫엯븯뒗 Knock-in쓽 寃쎌슦뒗 CRISPR/Cas9 system씠 쑀쟾옄 꽌뿴 듅씠쟻쑝濡 諛섏쓳븯硫 target guide RNA瑜 꽑젙빐빞 븯湲 븣臾몄뿉 렪吏묓븯怨좎옄 븯뒗 쑀쟾옄 궡뿉 뵒옄씤븷 닔 엳뒗 遺쐞媛 留ㅼ슦 븳젙쟻씤 寃쎌슦 몴以솕 맂 諛⑸쾿쓣 쟻슜븯湲 뼱졄떎. 蹂 뿰援ъ뿉꽌뒗 븯굹쓽 target guide RNA瑜 궗슜븯뒗 寃쎌슦, in vitro뿉꽌 RNA瑜 transcription 떆궓 sgRNA 삎깭濡 二쇱엯븳 寃곌낵뿉꽌뒗 Cas9 떒諛깆쭏쓽 냽룄 긽愿뾾씠 理쒖쥌 쑀쟾옄 궫엯쓣 솗씤븷 닔 뾾뿀떎(Table 3). 諛섎㈃뿉 target guide RNA瑜 crRAN/tracrRNA 삎깭濡 Cas9 떒諛깆쭏怨 injection븳 寃쎌슦, Cas9 떒諛깆쭏쓣 怨좊냽룄(100 ng/μL)濡 궗슜뻽쓣 븣蹂대떎 냽룄(10 ng/μL)濡 궗슜뻽쓣 븣 슚怨쇱쟻쑝濡 쑀쟾옄媛 궫엯릺뒗(4.1% vs. 1.7%) 寃껋쓣 솗씤븯떎. 쑀쟾옄 궫엯 留덉슦뒪 媛숈 삎吏덉쟾솚 留덉슦뒪瑜 젣옉븿뿉 엳뼱 以묒슂븳 슂씤쑝濡 옉슜븯뒗 寃껋씠 microinjection 湲곗닠씠떎. 蹂 뿰援ы뿉꽌 젣옉뻽뜕 쑀쟾옄瑜 怨쇰컻쁽 떆궓 삎吏덉쟾솚(transgenic, Tg) 留덉슦뒪쓽 microinjection 꽦怨듬쪧 20~50%쓽 넂 Tg 꽦怨듬쪧쓣 蹂댁떎(data not shown). 븯吏留 씠踰 site-specific hIgG Lc KI 留덉슦뒪 깮궛뿉꽌뒗 理쒖쥌쟻쑝濡 KI 꽦怨듬쪧씠 4.1%뿉 洹몄낀떎. 씠윭븳 李⑥씠뒗 micro-injection쓽 湲곗닠쟻 븳怨꾨씪湲곕낫떎뒗 쑀쟾옄 꽌뿴쓽 듅꽦쑝濡 뙋떒븷 닔 엳떎.

蹂 뿰援щ 넻빐 젣옉맂 hIgG Lc KI 留덉슦뒪 異뷀썑 젣옉븷 IgG heavy chain쓽 媛蹂 쁺뿭쓣 吏땶 留덉슦뒪瑜 넻빐 궗엺쓽 빆泥대 깮궛븯뒗 留덉슦뒪瑜 理쒖쥌쟻쑝濡 깮궛븯怨좎옄 븳떎. 씠踰 뿰援щ뒗 뼢썑 빆泥 移섎즺젣 媛쒕컻 諛 뿰援ъ뿉 留롮 솢슜 媛移섍 엳쓣 寃껋쑝濡 湲곕맂떎.

ACKNOWLEDGEMENT

蹂 끉臾몄 2019뀈룄 떊븳븰援 븰닠뿰援щ퉬 吏썝쑝濡 뿰援щ릺뿀쓬.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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