Special Topic: Stem Cells

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1 Special Topic: Stem Cells Review Progress in Biochemistry and Biophysics 2011, 38(11): 995~1000 wwwpibbaccn * ( ) (ES) ES ES ES ES Q813 Q789 DOI: /SPJ Brown-Norway Wistar ES DA SD (embryonic stem cells ES cells) ES Wistar ES (gene knockout) SD Wistar DA ES DA2 DA8 [2 4] 2004 ES [5] ES 121 ES ES ES ES 1 ES 11 ES 12 ES ES ES 2008 ES ES [1] Dark Agouti (DA) Fischer 344 (F344) Sprague-Dawley (SD) LIF 2003 Ying [6] (BMP) LIF ES ES ES * Tel: , mengshu@moonibpaccn

2 996 Prog Biochem Biophys 2011; 38 (11) ES FGF/MAPK/ERK ES 3i/2i GSK3 ES 3i 3 ( 1) ES CHIR99021 PD SU5402 N2B27 CHIR (GSK3) PD SU5402 2i/3i ES MAPK FGFR ES PD PD SU5402 MAPK ES [1 2, 7 8] 3i 2i ES PD (3i) SU5402 CHIR99021 PD (2i) CHIR99021 MAPK FGFR GSK3 MAPK FGFR GSK3 ES ES Fig 1 The ground state of rat ES cell self-renewal under N2B27 medium supplemented with small inhibitor [8] 1 [8] N2B27 ES MAPK: ; FGFR: ; GSK3: [4] 3i ES ES (integrin) 13 ES ES integrin ES integrinα7 α6 (AKP) ES (embryoid C3H/An body EB) ES L ES L 2i oct4 Sox2 MEF/3i ES Nanog SSEA-1 [2] SSEA-2 TRA-1-60 TRA-1-8l ES ES ( 1) ES ES 2i ES 3 (embryonic body EB)

3 2011; 38 (11) 997 β -tubulin DA2 DA8 ES [7] myosin Gata4 ES [5, 9] DA ES F344 Table 1 Difference in the common markers of human, mouse, and rat ES cells 1 ES hes mes res SSEA SSEA N/A SSEA TRA TRA N/A Oct Sox Nanog AP N/A Kitada [12] (sleeping beauty SB) ENU Robo2 ZFN ES SB GFP 2010 ES Izsvak [13] 21 ENU 2003 Zan [10] (ENU) ENU DNA Brca1 Brca2 23 (ZFN) 2007 (zinc finger nuclease ZFN) Homberg [11] ENU DNA 5-5- DNA ENU DNA 2009 Geurts [14] ZFN

4 998 Prog Biochem Biophys 2011; 38 (11) ZFN DNA mrna 2010 Mashimo [15] ES ZFN mrna IL-2 γ X (X-SCID) ZFN ( 6 ) ES PCR ZFN ZFN (off-target effect) ES Southern blot [14 15] (>1 kb) 24 ES ES ES 2008 ES ES 2010 ES p53 ES [1, 4] ES Fig 2 Flowchart outlining how to generate gene knockout rats [9] [9] 2 a β (CAG) 2011 Huang [8] p53 (PGK) p53 ES 14 p53 -/- PGK 120 ES b A p53 (DTA) (71%) 6 DTA ES DTA 256% p53 +/- ES p53 +/- c DA p53 -/- (BAC) DA [4] DNA Ying DNA ES p53 p53 ( 2)

5 2011; 38 (11) 999 Cre/LoxP Cre 4 Cre ROSA26 25~30 90% BRCA1 BRCA2, 3 ES ES ES Cell ( ES FOXP1 [16] ) TAF3 [17] ES ES [18] [1] Buehr M, Meek S, Blair K, ES 3 et al Capture of authentic embryonic stem cells from rat blastocysts Cell, 2008, 135(7): [2] Li P, Tong C, Mehrian R, et al Germline competent embryonic BMP Activin mtor stem cells derived from rat blastocysts Cell, 2008, 135(7): 1299 ES 1310 ES at high efficiency Mol Reprod Dev, 2010, 77(2): 94 Brown Norway [19] NB 2010, 467(7312): ES [20] Harbor Laboratory Press, 2003 ES ES MAPK P53 2i-LIF ES stem cell self-renewal Nature, 2008, 453(7194): [21] ES 2011, 10(7): ES Protocols, 2011, 6(6): [3] Hirabayashi M, Kato M, Kobavashi T, et al Establishment of rat embryonic stem cell lines that can participate in germline chimerae [4] Tong C, Li P, Wu N L, et al Production of p53 gene knockout rats by homologous recombination in embryonic stem cells Nature, [5] Nagy A, Gertsenstein M, Vintersten K, et al Manipulating the Mouse Embryo, A Laboratory Manual 3rd New York: Cold Spring [6] Ying Q L, Nichols J, Chambers I, et al BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3 Cell, 2003, 115 (3): [7] Ying Q L, Wray J, Nichols J, et al The ground state of embryonic [8] Huang G, Tong C, Kumbhani D S, et al Beyond knockout rats: New insights into finer genome manipulation in rats Cell Cycle, [9] Tong C, Huang G, Ashton C, et al Generating gene knockout rats by homologous recombination in embryonic stem cells Nature

6 1000 Prog Biochem Biophys 2011; 38 (11) [10] Zan Y, Haag D J, Chen K S, et al Production of knockout rats using ENU mutagenesis and a yeast-based screening assay Nat Biotechnol, 2003, 21(6): [11] Homberg J R, Olivier J D, Smits B M, et al Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system Neuroscience, 2007, 146(4): [12] Kitada K, Ishishita S, et al Transposon-tagged mutagenesis in the rat Nat Methods, 2007, 4(2): [13] Izsvak Z, Frohlich J, Grabundzija I, et al Generating knockout rats by transposon mutagenesis in spermatogonial stem cells Nat Methods, 2010, 7(6): [14] Geurts A M, Cost G J, Freyvert Y, et al Knockout rats via embryo microinjection of zinc-finger nucleases Science, 2009, 325(5939): 433 [15] Mashimo T, Takizawa A, Voigt B, et al Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases PLoS One, 2010, 5(1): e8870 [16] Gabut M, Samavarchi-Tehrani P, Wang X, et al An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming Cell, 2011, 147(1): [17] Liu Z, Scannell D R, Eisen M B, et al Control of embryonic stem cell lineage commitment by core promoter factor, TAF3 Cell, 2011, 146(5): [18] Li C, Yang Y, Gu J, et al Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts Cell Res, 2009, 19(2): [19] Zhao X, Lv Z, Liu L, et al Derivation of embryonic stem cells from brown norway rats blastocysts J Genet Genomics, 2010, 37 (7): [20] Shen Y, Shi C, Wei W, et al The heterogeneity and dynamic equilibrium of rat embryonic stem cells Cell Res, 2011, 21 (7): [21] Cao N, Liao J, Liu Z, et al In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes Cell Res, 2011, 21 (9): Progress in The Genetic Manipulation Technology of Embryonic Stem Cells in Rats MENG Shu * (The Core Facility for Protein Research, Institute of Biophysics, The Chinese Academy of Sciences, Beijing , China) Abstract The successful establishment of rat ES cell lines enables possible genetic manipulation in rats Using homologous recombination to modify ES cell genes provides a foundation to temporal and tissue-specific knockout rats The author introduces the establishment process of rat ES cell, summarizes its culture and identification skills, and analyzes the weakness and strength of various rat knockout techniques In the context of increasing research on stem cell, knockout technology based on rat ES cells, which allows the most efficient modification of specific genes, shall contribute more to elucidate gene function, dissect the genetic mechanisms of human diseases and identify potential targets for drug development Key words ES cell, genetic manipulation, gene knockout, stem cell DOI: /SPJ *Corresponding author Tel: , mengshu@moonibpaccn Received: October 19, 2011 Accepted: October 23, 2011