Strategies to Optimize the Expression of Mammary Gland Bioreactor

doi:10.13560/j.cnki.biotech.bull.1985.2022-1150

Abstract

Abstract:

Mammary gland bioreactor refers to the technology of importing foreign genes into animal genomes which express specifically in animal mammary gland, and using the function of synthesizing and secreting proteins of mammary glands to obtain recombinant proteins in the milk. Mammary gland bioreactor is regarded as a technological innovation for the production of medicinal protein and nutritional protein in the bio-pharmaceutical industry due to its advantages of high expression, low cost and synthesized protein being close to natural proteins in structure. However, due to the random integration of foreign genes and unstable expression of recombinant proteins, its application is largely limited. Based on the current development status of mammary gland bioreactor, this paper reviewed the optimization strategies of mammary gland bioreactor from three perspectives: using gene editing technology, screening suitable foreign gene integration sites and improving the regulatory sequence of exogenous proteins, so as to provide theoretical reference for improving the expression of recombinant protein produced by mammary gland bioreactor

Key words: mammary gland bioreactor, chromatin accessibility, site-specific integration, CRISPR/Cas9, regulatory element, target site

LIU Xiao-yan, ZHU Zhen-liang, SHI Guang-yu, HUA Zi-yu, YANG Chen, ZHANG Yong, LIU Jun. Strategies to Optimize the Expression of Mammary Gland Bioreactor[J]. Biotechnology Bulletin, 2023, 39(5): 77-91.

Figures/Tables 3

References 135

[3]	Qian X, Zhao FQ. Current major advances in the regulation of milk protein gene expression[J]. Crit Rev Eukaryot Gene Expr, 2014, 24(4): 357-378. doi: 10.1615/CritRevEukaryotGeneExpr.v24.i4 URL
[4]	Clark AJ. The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins[J]. J Mammary Gland Biol Neoplasia, 1998, 3(3): 337-350. doi: 10.1023/A:1018723712996 URL
[5]	Lu D, Liu S, Ding FR, et al. Large-scale production of functional human lysozyme from marker-free transgenic cloned cows[J]. Sci Rep, 2016, 6: 22947. doi: 10.1038/srep22947 pmid: 26961596
[6]	Cui CC, Song YJ, Liu J, et al. Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk[J]. Sci Rep, 2015, 5: 10482. doi: 10.1038/srep10482 pmid: 25994151
[7]	Luo Y, Wang YS, Liu J, et al. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands[J]. Sci Rep, 2016, 6: 20657. doi: 10.1038/srep20657 pmid: 26853907
[8]	McCreath KJ, Howcroft J, Campbell KH, et al. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells[J]. Nature, 2000, 405(6790): 1066-1069. doi: 10.1038/35016604
[9]	Song SZ, Ge X, Cheng YB, et al. High-level expression of a novel recombinant human plasminogen activator(rhPA)in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro[J]. Mol Biol Rep, 2016, 43(8): 775-783. doi: 10.1007/s11033-016-4020-0 URL
[10]	Wang Q, Hao S, Ma L, et al. Comparison of human coagulation factor VIII expression directed by Cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice[J]. Blood Coagul Fibrinolysis, 2015, 26(7): 755-761. doi: 10.1097/MBC.0000000000000318 URL
[11]	Gil GC, Velander WH, van Cott KE. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk[J]. Glycobiology, 2008, 18(7): 526-539. doi: 10.1093/glycob/cwn035 URL
[12]	成勇, 王玉阁, 罗金平. 由成年转基因山羊体细胞而来的克隆山羊[J]. 生物工程学报, 2002, 18(1): 79-83.
	Cheng Y, Wang YG, Luo JP, et al. Cloned goats produced from the somatic cells of an adult transgenic goat[J]. Chinese Journal of Biotechnology, 2002, 18(1): 79-83. pmid: 11977606
[13]	Koles K, Pieper FR, et al. N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits[J]. Glycobiology, 2004, 14(1): 51-64. pmid: 14514717
[14]	Bijvoet AG, Kroos MA, Pieper FR, et al. Expression of cDNA-encoded human acid alpha-glucosidase in milk of transgenic mice[J]. Biochim Biophys Acta, 1996, 1308(2): 93-96. pmid: 8764823
[15]	Uusi-Oukari M, Hyttinen JM, Korhonen VP, et al. Bovine alpha s1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice[J]. Transgenic Res, 1997, 6(1): 75-84. pmid: 9032980
[16]	Edmunds T, van Patten SM, Pollock J, et al. Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin[J]. Blood, 1998, 91(12): 4561-4571. pmid: 9616152
[17]	Meade H, Gates L, Lacy E, et al. Bovine AlphaS1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J]. Nature Biotechnology, 1990, 8(5): 443-446. pmid: 1369989
[18]	Brem G, Hartl P, Besenfelder U, et al. Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1(IGF-1)in the mammary gland of transgenic rabbits[J]. Gene, 1994, 149(2): 351-355. pmid: 7959016
[19]	Lu R, Zhang T, Wu DJ, et al. Production of functional human CuZn-SOD and EC-SOD in bitransgenic cloned goat milk[J]. Transgenic Res, 2018, 27(4): 343-354. doi: 10.1007/s11248-018-0080-3 pmid: 29926349
[20]	Hua RM, Liu JX, Li Y, et al. Novel functional recombinant human follicle-stimulating hormone acquired from goat milk[J]. J Agric Food Chem, 2021, 69(9): 2793-2804. doi: 10.1021/acs.jafc.0c07208 URL
[21]	Ma T, Tao J, Yang M, et al. An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep[J]. J Pineal Res, 2017, 63(1): 2017 Aug; 63(1).
[22]	Tavares KC, Dias AC, Lazzarotto CR, et al. Transient expression of functional glucocerebrosidase for treatment of Gaucher’s disease in the goat mammary gland[J]. Mol Biotechnol, 2016, 58(1): 47-55. doi: 10.1007/s12033-015-9902-1 URL
[23]	Morcöl T, Akers RM, Johnson JL, et al. The porcine mammary gland as a bioreactor for complex proteins[J]. Ann N Y Acad Sci, 1994, 721: 218-233. doi: 10.1111/nyas.1994.721.issue-1 URL
[24]	Gong GH, Zhang W, Xie LP, et al. Expression of a recombinant anti-programed cell death 1 antibody in the mammary gland of transgenic mice[J]. Prep Biochem Biotechnol, 2021, 51(2): 183-190. doi: 10.1080/10826068.2020.1805755 URL
[25]	Baldassarre H, Hockley DK, Olaniyan B, et al. Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland[J]. Transgenic Res, 2008, 17(5): 863-872. doi: 10.1007/s11248-008-9184-5 pmid: 18483775
[26]	Lipinski D, Zeyland J, Szalata M, et al. Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q[J]. J Appl Genet, 2012, 53(4): 435-442. doi: 10.1007/s13353-012-0110-4 pmid: 22898896
[27]	Bulleid NJ, John DC, Kadler KE. Recombinant expression systems for the production of collagen[J]. Biochem Soc Trans, 2000, 28(4): 350-353. doi: 10.1042/bst0280350 URL
[28]	Tang B, Yu SS, Zheng M, et al. High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice[J]. Transgenic Res, 2008, 17(4): 727-732. doi: 10.1007/s11248-007-9162-3 pmid: 18183493
[29]	Yu HQ, Wang XB, Zhu L, et al. Establishment of a rapid and scalable gene expression system in livestock by site-specific integration[J]. Gene, 2013, 515(2): 367-371. doi: 10.1016/j.gene.2012.10.017 pmid: 23089494
[30]	Ahmadi M, Damavandi N, Akbari Eidgahi MR, et al. Utilization of site-specific recombination in biopharmaceutical production[J]. Iran Biomed J, 2016, 20(2): 68-76. pmid: 26602035
[31]	Jensen KT, Fløe L, Petersen TS, et al. Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency[J]. FEBS Lett, 2017, 591(13): 1892-1901. doi: 10.1002/1873-3468.12707 pmid: 28580607
[32]	Barnes LM, Bentley CM, Dickson AJ. Stability of protein production from recombinant mammalian cells[J]. Biotechnol Bioeng, 2003, 81(6): 631-639. pmid: 12529877
[33]	Houdebine LM. Production of pharmaceutical proteins by transgenic animals[J]. Comp Immunol Microbiol Infect Dis, 2009, 32(2): 107-121. doi: 10.1016/j.cimid.2007.11.005 URL
[34]	Wang TY, Guo X. Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems[J]. Appl Microbiol Biotechnol, 2020, 104(13): 5673-5688. doi: 10.1007/s00253-020-10640-w
[35]	Shepelev MV, Kalinichenko SV, Deykin AV, et al. Production of recombinant proteins in the milk of transgenic animals: current state and prospects[J]. Acta Naturae, 2018, 10(3): 40-47. pmid: 30397525
[36]	Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system[J]. Nat Protoc, 2013, 8(11): 2281-2308. doi: 10.1038/nprot.2013.143 pmid: 24157548
[37]	Pavani G, Amendola M. Targeted gene delivery: where to land[J]. Front Genome Ed, 2021, 2: 609650. doi: 10.3389/fgeed.2020.609650 URL
[38]	Kumar S, Clarke AR, Hooper ML, et al. Milk composition and lactation of beta-casein-deficient mice[J]. PNAS, 1994, 91(13): 6138-6142. pmid: 8016126
[39]	宋绍征, 张婷, 潘生强, 等. CRISPR/Cas9系统介导的人乳铁蛋白基因在山羊β-乳球蛋白基因座定点敲入[J]. 中国农业大学学报, 2020, 25(7): 111-119.
	Song SZ, Zhang T, Pan SQ, et al. hLF gene knock-in at the BLGlocus of goat by CRISPR/Cas9 system[J]. J China Agric Univ, 2020, 25(7): 111-119.
[40]	冀艳华, 徐乔璐, 刘军, 等. TALEN介导猪瘟病毒结构蛋白基因E0敲入山羊乳腺上皮细胞β-乳球蛋白基因座[J]. 中国兽医学报, 2017, 37(7): 1206-1211.
	Ji YH, Xu QL, Liu J, et al. CSFV E0 gene knocked-in β-lactoglobulin locus in goat mammary epithelial cells by TALEN[J]. Chin J Vet Sci, 2017, 37(7): 1206-1211.
[41]	An LY, Yang L, Huang YJ, et al. Generating goat mammary gland bioreactors for producing recombinant proteins by gene targeting[J]. Methods Mol Biol, 2019, 1874: 391-401. doi: 10.1007/978-1-4939-8831-0_23 pmid: 30353527
[42]	Wu MM, Wei CH, Lian ZX, et al. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system[J]. Sci Rep, 2016, 6: 24360. doi: 10.1038/srep24360 pmid: 27063570
[43]	Li XP, Yang Y, Bu L, et al. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing[J]. Cell Res, 2014, 24(4): 501-504. doi: 10.1038/cr.2014.15 pmid: 24503648
[44]	Yuan MK, Zhang JC, Gao YP, et al. HMEJ-based safe-harbor genome editing enables efficient generation of cattle with increased resistance to tuberculosis[J]. J Biol Chem, 2021, 296: 100497. doi: 10.1016/j.jbc.2021.100497 URL
[45]	Jeong YH, Kim YJ, Kim EY, et al. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination[J]. Zygote, 2016, 24(3): 442-456. doi: 10.1017/S0967199415000374 URL
[46]	Liu X, Wang YS, Guo WJ, et al. Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows[J]. Nat Commun, 2013, 4: 2565. doi: 10.1038/ncomms3565 pmid: 24121612
[47]	周文君, 郭日红, 邓明田, 等. RS-1提高CRISPR-Cas9系统介导的人乳铁蛋白基因敲入效率[J]. 生物工程学报, 2017, 33(8): 1224-1234.
	Zhou WJ, Guo RH, Deng MT, et al. RS-1 enhanced the efficiency of CRISPR-Cas9 mediated knock-in of human lactoferrin[J]. Chin J Biotechnol, 2017, 33(8): 1224-1234.
[48]	Hu SW, Hall J, Wang Z, et al. TALEN-mediated targeted insertion of transforming growth factor-beta 1(TGF-beta 1)gene into the goat AAVS1 locus[J]. Transgenic Research, 2014, 23(1): 203-204.
[49]	Ruan JX, Li HG, Xu K, et al. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs[J]. Sci Rep, 2015, 5: 14253. doi: 10.1038/srep14253 pmid: 26381350
[50]	Brady JR, Tan MC, Whittaker CA, et al. Identifying improved sites for heterologous gene integration using ATAC-seq[J]. ACS Synth Biol, 2020, 9(9): 2515-2524. doi: 10.1021/acssynbio.0c00299 pmid: 32786350
[51]	Iler N, Goodwin AJ, McInerney J, et al. Targeted remodeling of human beta-globin promoter chromatin structure produces increased expression and decreased silencing[J]. Blood Cells Mol Dis, 1999, 25(1): 47-60. doi: 10.1006/bcmd.1999.0226 URL
[52]	Nemeth MJ, Bodine DM, Garrett LJ, et al. An erythroid-specific chromatin opening element reorganizes beta-globin promoter chromatin structure and augments gene expression[J]. Blood Cells Mol Dis, 2001, 27(4): 767-780. doi: 10.1006/bcmd.2001.0448 URL
[53]	Eyquem J, Poirot L, Galetto R, et al. Characterization of three loci for homologous gene targeting and transgene expression[J]. Biotechnol Bioeng, 2013, 110(8): 2225-2235. doi: 10.1002/bit.24892 pmid: 23475535
[54]	Yan CH, Boyd DD. Histone H 3 acetylation and H3 K4 methylation define distinct chromatin regions permissive for transgene expression[J]. Mol Cell Biol, 2006, 26(17): 6357-6371. doi: 10.1128/MCB.00311-06 URL
[55]	Veith N, Ziehr H, MacLeod RAF, et al. Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary(CHO)cell lines[J]. BMC Biotechnol, 2016, 16: 6. doi: 10.1186/s12896-016-0238-0 URL
[56]	Singh K, Erdman RA, Swanson KM, et al. Epigenetic regulation of milk production in dairy cows[J]. J Mammary Gland Biol Neoplasia, 2010, 15(1): 101-112. doi: 10.1007/s10911-010-9164-2 URL
[57]	Clapier CR, Iwasa J, Cairns BR, et al. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes[J]. Nat Rev Mol Cell Biol, 2017, 18(7): 407-422. doi: 10.1038/nrm.2017.26 URL
[58]	Zhao YX, Hou Y, Xu YY, et al. A compendium and comparative epigenomics analysis of Cis-regulatory elements in the pig genome[J]. Nat Commun, 2021, 12(1): 2217. doi: 10.1038/s41467-021-22448-x
[1]	O'Flaherty R, Bergin A, Flampouri E, et al. Mammalian cell culture for production of recombinant proteins: a review of the critical steps in their biomanufacturing[J]. Biotechnol Adv, 2020, 43: 107552. doi: 10.1016/j.biotechadv.2020.107552 URL
[2]	Wang YL, Zhao SH, Bai L, et al. Expression systems and species used for transgenic animal bioreactors[J]. Biomed Res Int, 2013, 2013: 580463.
[59]	Reilly SK, Yin J, Ayoub AE, et al. Evolutionary genomics. Evolutionary changes in promoter and enhancer activity during human corticogenesis[J]. Science, 2015, 347(6226): 1155-1159. doi: 10.1126/science.1260943 pmid: 25745175
[60]	Chen KF, Chen Z, Wu DY, et al. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes[J]. Nat Genet, 2015, 47(10): 1149-1157. doi: 10.1038/ng.3385 pmid: 26301496
[61]	Bartosovic M, Kabbe M, Castelo-Branco G. Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues[J]. Nat Biotechnol, 2021, 39(7): 825-835. doi: 10.1038/s41587-021-00869-9 pmid: 33846645
[62]	Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: from properties to genome-wide predictions[J]. Nat Rev Genet, 2014, 15(4): 272-286. doi: 10.1038/nrg3682 pmid: 24614317
[63]	Rijnkels M, Freeman-Zadrowski C, Hernandez J, et al. Epigenetic modifications unlock the milk protein gene loci during mouse mammary gland development and differentiation[J]. PLoS One, 2013, 8(1): e53270. doi: 10.1371/journal.pone.0053270 URL
[64]	Singh K, Molenaar AJ, Swanson KM, et al. Epigenetics: a possible role in acute and transgenerational regulation of dairy cow milk production[J]. Animal, 2012, 6(3): 375-381. doi: 10.1017/S1751731111002564 pmid: 22436216
[65]	Zhang XY, Zhang SH, Ma L, et al. Reduced representation bisulfite sequencing(RRBS)of dairy goat mammary glands reveals DNA methylation profiles of integrated genome-wide and critical milk-related genes[J]. Oncotarget, 2017, 8(70): 115326-115344. doi: 10.18632/oncotarget.v8i70 URL
[66]	Yan F, Powell DR, Curtis DJ, et al. From reads to insight: a hitchhiker's guide to ATAC-seq data analysis[J]. Genome Biol, 2020, 21(1): 22. doi: 10.1186/s13059-020-1929-3 pmid: 32014034
[67]	Papapetrou EP, Schambach A. Gene insertion into genomic safe harbors for human gene therapy[J]. Mol Ther, 2016, 24(4): 678-684. doi: 10.1038/mt.2016.38 pmid: 26867951
[68]	Sadelain M, Papapetrou EP, Bushman FD. Safe harbours for the integration of new DNA in the human genome[J]. Nat Rev Cancer, 2011, 12(1): 51-58. doi: 10.1038/nrc3179 pmid: 22129804
[69]	Pellenz S, Phelps M, Tang WL, et al. New human chromosomal sites with “safe harbor” potential for targeted transgene insertion[J]. Hum Gene Ther, 2019, 30(7): 814-828. doi: 10.1089/hum.2018.169 pmid: 30793977
[70]	Beagan JA, Phillips-Cremins JE. On the existence and functionality of topologically associating domains[J]. Nat Genet, 2020, 52(1): 8-16. doi: 10.1038/s41588-019-0561-1 pmid: 31925403
[71]	Hilliard W, Lee KH. Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis[J]. Biotechnol Bioeng, 2021, 118(2): 659-675. doi: 10.1002/bit.27599 pmid: 33049068
[72]	Meuleman W, Muratov A, Rynes E, et al. Index and biological spectrum of human DNase I hypersensitive sites[J]. Nature, 2020, 584(7820): 244-251. doi: 10.1038/s41586-020-2559-3
[73]	Autio MI, Motakis E, Perrin A, et al. Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells[J]. bioRxiv, 2022, DOI:10.1101/2021.12.07.471422. doi: 10.1101/2021.12.07.471422
[74]	Bhagwan JR, Collins E, Mosqueira D, et al. Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour[J]. F1000Research, 2019, 8: 1911. doi: 10.12688/f1000research.19894.2 pmid: 32789000
[75]	Yu HQ, Chen JQ, Liu SG, et al. Large-scale production of functional human lysozyme in transgenic cloned goats[J]. J Biotechnol, 2013, 168(4): 676-683. pmid: 24432381
[76]	Zhang T, Yuan YG, Lu R, et al. The goat β-casein/CMV chimeric promoter drives the expression of hLF in transgenic goats produced by cell transgene microinjection[J]. Int J Mol Med, 2019, 44(6): 2057-2064. doi: 10.3892/ijmm.2019.4382 pmid: 31661123
[77]	Cheng Y, An LY, Yuan YG, et al. Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice[J]. Mol Reprod Dev, 2012, 79(8): 573-585. doi: 10.1002/mrd.22063 pmid: 22730016
[78]	Li GC, Shi WQ, Chen G, et al. Construction and in vivo evaluation of a mammary gland-specific expression vector for human lysozyme[J]. Plasmid, 2014, 76: 47-53. doi: 10.1016/j.plasmid.2014.09.004 pmid: 25280784
[79]	Xu DH, Wang XY, Jia YL, et al. SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells[J]. J Cell Mol Med, 2018, 22(4): 2231-2239. doi: 10.1111/jcmm.2018.22.issue-4 URL
[80]	Lu D, Li QY, Wu ZB, et al. High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs[J]. PLoS One, 2014, 9(2): e89130. doi: 10.1371/journal.pone.0089130 URL
[81]	Luo Y, Liu J, Liu QQ, et al. Chicken hypersensitive site-4 insulator increases human serum albumin expression in bovine mammary epithelial cells modified with phiC31 integrase[J]. Biotechnol Lett, 2013, 35(4): 529-537. doi: 10.1007/s10529-012-1125-y pmid: 23264267
[82]	Naderi F, Hashemi M, Bayat H, et al. The augmenting effects of the tDNA insulator on stable expression of monoclonal antibody in Chinese hamster ovary cells[J]. Monoclon Antib Immunodiagn Immunother, 2018, 37(5): 200-206. doi: 10.1089/mab.2018.0015 pmid: 30362930
[83]	Zhang JH, Zhang JH, Wang XY, et al. Distance effect characteristic of the matrix attachment region increases recombinant protein expression in Chinese hamster ovary cells[J]. Biotechnol Lett, 2020, 42(2): 187-196. doi: 10.1007/s10529-019-02775-2
[84]	Girod PA, Zahn-Zabal M, Mermod N. Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines[J]. Biotechnol Bioeng, 2005, 91(1): 1-11. doi: 10.1002/(ISSN)1097-0290 URL
[85]	Kim JD, Yoon Y, Hwang HY, et al. Efficient selection of stable Chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon beta SAR element[J]. Biotechnol Prog, 2005, 21(3): 933-937. doi: 10.1021/(ISSN)1520-6033 URL
[86]	Kim JM, Kim JS, Park DH, et al. Improved recombinant gene expression in CHO cells using matrix attachment regions[J]. J Biotechnol, 2004, 107(2): 95-105. doi: 10.1016/j.jbiotec.2003.09.015 URL
[87]	Jia YL, Guo X, Wang XC, et al. Human genome-derived TOP1 matrix attachment region enhances transgene expression in the transfected CHO cells[J]. Biotechnol Lett, 2019, 41(6/7): 701-709. doi: 10.1007/s10529-019-02673-7
[88]	Tian ZW, Xu DH, Wang TY, et al. Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells[J]. J Cell Mol Med, 2018, 22(2): 1095-1102.
[89]	Benton T, Chen T, McEntee M, et al. The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein[J]. Cytotechnology, 2002, 38(1-3): 43-46. doi: 10.1023/A:1021141712344 pmid: 19003085
[90]	Saunders F, Sweeney B, Antoniou MN, et al. Chromatin function modifying elements in an industrial antibody production platform—comparison of UCOE, MAR, STAR and cHS4 elements[J]. PLoS One, 2015, 10(4): e0120096. doi: 10.1371/journal.pone.0120096 URL
[91]	Kwaks THJ, Barnett P, Hemrika W, et al. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells[J]. Nat Biotechnol, 2003, 21(5): 553-558. pmid: 12679786
[92]	Maksimenko OG, Deykin AV, Khodarovich YM, et al. Use of transgenic animals in biotechnology: prospects and problems[J]. Acta Naturae, 2013, 5(1): 33-46. pmid: 23556129
[93]	Li S, Huang SH, Qiao SY, et al. Cloning and functional characterization of STAT5a and STAT5b genes in buffalo mammary epithelial cells[J]. Anim Biotechnol, 2020, 31(1): 59-66. doi: 10.1080/10495398.2018.1538014 pmid: 30431388
[94]	Qian X, Zhao FQ. Regulatory roles of Oct proteins in the mammary gland[J]. Biochim Biophys Acta, 2016, 1859(6): 812-819. doi: 10.1016/j.bbagrm.2016.03.015 pmid: 27044595
[95]	Song N, Luo J, Huang L, et al. Mutation of signal transducer and activator of transcription 5 (STAT5) binding sites decreases milk allergen αS1-casein content in goat mammary epithelial cells[J]. Foods, 2022, 11(3): 346. doi: 10.3390/foods11030346 URL
[96]	Kung MH, Lee YJ, Hsu JT, et al. A functional study of proximal goat β-casein promoter and intron 1 in immortalized goat mammary epithelial cells[J]. J Dairy Sci, 2015, 98(6): 3859-3875. doi: 10.3168/jds.2014-9054 pmid: 25841968
[97]	Panigrahi A, O'Malley BW. Mechanisms of enhancer action: the known and the unknown[J]. Genome Biol, 2021, 22(1): 108. doi: 10.1186/s13059-021-02322-1 pmid: 33858480
[98]	Galouzis CC, Furlong EEM. Regulating specificity in enhancer-promoter communication[J]. Curr Opin Cell Biol, 2022, 75: 102065. doi: 10.1016/j.ceb.2022.01.010 URL
[99]	Osterwalder M, Barozzi I, Tissières V, et al. Enhancer redundancy provides phenotypic robustness in mammalian development[J]. Nature, 2018, 554(7691): 239-243. doi: 10.1038/nature25461 URL
[100]	Song W, Sharan R, Ovcharenko I. The first enhancer in an enhancer chain safeguards subsequent enhancer-promoter contacts.
[101]	Shin HY, Willi M, HyunYoo K, et al. Hierarchy within the mammary STAT5-driven wap super-enhancer[J]. Nat Genet, 2016, 48(8): 904-911. doi: 10.1038/ng.3606 pmid: 27376239
[102]	Hnisz D, Abraham BJ, Lee TI, et al. Super-enhancers in the control of cell identity and disease[J]. Cell, 2013, 155(4): 934-947. doi: 10.1016/j.cell.2013.09.053 pmid: 24119843
[103]	Lee Z, Raabe M, Hu WS. Epigenomic features revealed by ATAC-seq impact transgene expression in CHO cells[J]. Biotechnol Bioeng, 2021, 118(5): 1851-1861. doi: 10.1002/bit.27701 pmid: 33521928
[104]	Zeng XK, Lee HK, Wang CC, et al. The interdependence of mammary-specific super-enhancers and their native promoters facilitates gene activation during pregnancy[J]. Exp Mol Med, 2020, 52(4): 682-690. doi: 10.1038/s12276-020-0425-x pmid: 32321991
[105]	Borsari B, Villegas-Mirón P, Pérez-Lluch S, et al. Enhancers with tissue-specific activity are enriched in intronic regions[J]. Genome Res, 2021, 31(8): 1325-1336. doi: 10.1101/gr.270371.120 pmid: 34290042
[106]	Andersson R, Sandelin A. Determinants of enhancer and promoter activities of regulatory elements[J]. Nat Rev Genet, 2020, 21(2): 71-87. doi: 10.1038/s41576-019-0173-8 pmid: 31605096
[107]	Corrales M, Rosado A, Cortini R, et al. Clustering of Drosophila housekeeping promoters facilitates their expression[J]. Genome Res, 2017, 27(7): 1153-1161. doi: 10.1101/gr.211433.116 pmid: 28420691
[108]	Zhu I, Song W, Ovcharenko I, et al. A model of active transcription hubs that unifies the roles of active promoters and enhancers[J]. Nucleic Acids Res, 2021, 49(8): 4493-4505. doi: 10.1093/nar/gkab235 URL
[109]	Dong WH, Li CP, Yang Y, et al. Increasing transgenic expression in recombinant Chinese hamster ovary cells using introns in different directions[J]. Sheng Wu Gong Cheng Xue Bao, 2019, 35(6): 1071-1078.
[110]	Chorev M, Carmel L. The function of introns[J]. Front Genet, 2012, 3: 55. doi: 10.3389/fgene.2012.00055 pmid: 22518112
[111]	Bieberstein NI, Carrillo Oesterreich F, Straube K, et al. First exon length controls active chromatin signatures and transcription[J]. Cell Rep, 2012, 2(1): 62-68. doi: 10.1016/j.celrep.2012.05.019 pmid: 22840397
[112]	Agarwal N, Ansari A. Enhancement of transcription by a splicing-competent intron is dependent on promoter directionality[J]. PLoS Genet, 2016, 12(5): e1006047. doi: 10.1371/journal.pgen.1006047 URL
[113]	Palazzo AF, Mahadevan K, Tarnawsky SP. ALREX-elements and introns: two identity elements that promote mRNA nuclear export[J]. Wiley Interdiscip Rev RNA, 2013, 4(5): 523-533. doi: 10.1002/wrna.2013.4.issue-5 URL
[114]	Shaul O. How introns enhance gene expression[J]. Int J Biochem Cell Biol, 2017, 91(Pt B): 145-155. doi: S1357-2725(17)30154-1 pmid: 28673892
[115]	Houdebine LM. Design of vectors for optimizing transgene expression[M]// Transgenic Animal Technology. Amsterdam: Elsevier, 2014: 489-511.
[116]	Qu GS, Piazza CL, Smith D, et al. Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting[J]. eLife, 2018, 7: e34268. doi: 10.7554/eLife.34268 URL
[117]	Rose AB. Introns as gene regulators: a brick on the accelerator[J]. Front Genet, 2019, 9: 672. doi: 10.3389/fgene.2018.00672
[118]	Gallegos JE, Rose AB. The enduring mystery of intron-mediated enhancement[J]. Plant Sci, 2015, 237: 8-15. doi: 10.1016/j.plantsci.2015.04.017 pmid: 26089147
[119]	Dwyer K, Agarwal N, Gega A, et al. Proximity to the promoter and Terminator regions regulates the transcription enhancement potential of an intron[J]. Front Mol Biosci, 2021, 8: 712639. doi: 10.3389/fmolb.2021.712639 URL
[120]	Romanova N, Noll T. Engineered and natural promoters and chromatin-modifying elements for recombinant protein expression in CHO cells[J]. Biotechnol J, 2018, 13(3): e1700232.
[121]	Reddi PP, Urekar CJ, Abhyankar MM, et al. Role of an insulator in testis-specific gene transcription[J]. Ann N Y Acad Sci, 2007, 1120: 95-103. doi: 10.1196/annals.1411.012 URL
[122]	Chetverina D, Aoki T, Erokhin M, et al. Making connections: insulators organize eukaryotic chromosomes into independent cis-regulatory networks[J]. Bioessays, 2014, 36(2): 163-172. doi: 10.1002/bies.201300125 pmid: 24277632
[123]	Lu XB, Guo YH, Huang W. Characterization of the cHS4 insulator in mouse embryonic stem cells[J]. FEBS Open Bio, 2020, 10(4): 644-656. doi: 10.1002/feb4.v10.4 URL
[124]	Kisseljova NP, Dmitriev P, Katargin A, et al. DNA polymorphism and epigenetic marks modulate the affinity of a scaffold/matrix attachment region to the nuclear matrix[J]. Eur J Hum Genet, 2014, 22(9): 1117-1123. doi: 10.1038/ejhg.2013.306 pmid: 24448543
[125]	Guo X, Wang C, Wang TY. Chromatin-modifying elements for recombinant protein production in mammalian cell systems[J]. Crit Rev Biotechnol, 2020, 40(7): 1035-1043. doi: 10.1080/07388551.2020.1805401 pmid: 32777953
[126]	Harraghy N, Calabrese D, Fisch I, et al. Epigenetic regulatory elements: recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells[J]. Biotechnol J, 2015, 10(7): 967-978. doi: 10.1002/biot.201400649 pmid: 26099730
[127]	Zhao CP, Guo X, Chen SJ, et al. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells[J]. Sci Rep, 2017, 7: 42805. doi: 10.1038/srep42805
[128]	Nematpour F, Mahboudi F, Vaziri B, et al. Evaluating the expression profile and stability of different UCOE containing vector combinations in mAb-producing CHO cells[J]. BMC Biotechnol, 2017, 17(1): 18. doi: 10.1186/s12896-017-0330-0 pmid: 28228095
[129]	Rocha-Pizaña MDR, Ascencio-Favela G, Soto-García BM, et al. Evaluation of changes in promoters, use of UCOES and chain order to improve the antibody production in CHO cells[J]. Protein Expr Purif, 2017, 132: 108-115. doi: 10.1016/j.pep.2017.01.014 URL
[130]	Wang B, Guo Q, Liu LY, et al. Effect of interactions of chromatin regulatory elements with different promoters on the regulation of gene expression[J]. Chinese journal of biotechnology, 2021, 37(9): 3310-3322. doi: 10.13345/j.cjb.200748 pmid: 34622638
[131]	Mayr C. What are 3' UTRs doing?[J]. Cold Spring Harb Perspect Biol, 2019, 11(10): a034728. doi: 10.1101/cshperspect.a034728 URL
[132]	Kim JJ, Yu J, Bag J, et al. Translation attenuation via 3' terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and β-casein profile in milk[J]. RNA Biol, 2015, 12(3): 354-367. doi: 10.1080/15476286.2015.1017231 URL
[133]	Goodarzi H, Najafabadi HS, Oikonomou P, et al. Systematic discovery of structural elements governing stability of mammalian messenger RNAs[J]. Nature, 2012, 485(7397): 264-268. doi: 10.1038/nature11013
[134]	Cohen-Zontag O, Baez C, Lim LQJ, et al. A secretion-enhancing cis regulatory targeting element(SECReTE)involved in mRNA localization and protein synthesis[J]. PLoS Genet, 2019, 15(7): e1008248. doi: 10.1371/journal.pgen.1008248 URL
[135]	He Z, Song D, van Zalen S, et al. Structural determinants of human ζ-globin mRNA stability[J]. J Hematol Oncol, 2014, 7: 35. doi: 10.1186/1756-8722-7-35

重组蛋白 Recombinant protein	物种 Species	表达含量 Expression content /（μg·mL^-1）	时间/年 Time/Year	参考文献 References
溶菌酶Lysozym	奶牛Bovine	3 100	2016	[5]
人乳铁蛋白Human lactoferrin	山羊Capra hircus	3 200	2015	[6]
人血清白蛋白HSA	奶牛Bovine	2 500	2016	[7]
抗胰蛋白酶AAT	绵羊Ovis aries	650	2000	[8]
重组人纤溶酶原激活物 Recombinant human plasminogen activator	兔Leporidae	15.2-630	2016	[9]
凝血因子VIII FVlll	小鼠Mus musculus	1 000-4 000	2015	[10]
凝血因子IX FIX	猪Sus	100-400	2008	[11]
促红细胞生成素EPO	山羊Capra hircus	未报道Not reported	2002	[12]
人C1抑制剂Human C1 inhibitor	兔Leporidae	未报道Not reported	2003	[13]
人酸性α-葡萄糖苷酶 Human acid alpha-glucosidase	小鼠Mus musculus	1.5	1996	[14]
粒细胞巨噬细胞集落刺激因子GM-CSF	小鼠Mus musculus	200-4 600	1997	[15]
重组人抗凝血酶Recombinant human antithrombin	山羊Capra hircus	未报道Not reported	1998	[16]
人尿激酶Human urokinase	小鼠Mus musculus	1 000-2 000	1990	[17]
胰岛素样生长因子-1 IGF-1	兔Leporidae	1 000	1992	[18]
铜/锌超氧化物歧化酶、细胞外超氧化物歧化酶 CuZn-SOD、EC-SOD	山羊Capra hircus	100.14 ± 5.09、279.10 ± 5.38	2018	[19]
卵泡刺激素FSHα、FSHβ	山羊Capra hircus	0.28、0.30	2021	[20]
芳基烷基胺N-乙酰转移酶、乙酰5-羟色胺甲基转移酶AANAT、ASMT	绵羊Ovis aries	未报道Not reported	2017	[21]
葡糖脑苷脂酶Glucocerebrosidase	山羊Capra hircus	111.1 ± 8.1	2015	[22]
人蛋白C Human protein C	猪Sus	100-1 000	1994	[23]
抗程序性细胞死亡1抗体PD-1 antibody	小鼠Mus musculus	80.52 ± 0.82	2020	[24]
丁酰胆碱酯酶Butyrylcholinesterase	山羊Capra hircus	1 000-5 000	2008	[25]
生长激素Growth hormone	兔Leporidae	10	2012	[26]
胶原Collagen	小鼠Mus musculus	200	2000	[27]
抗CD20单克隆抗体Anti-CD20 mAB	小鼠Mus musculus	17	2008	[28]

重组蛋白 Recombinant protein	物种 Species	表达含量 Expression content /（μg·mL^-1）	时间/年 Time/Year	参考文献 References
溶菌酶Lysozym	奶牛Bovine	3 100	2016	[5]
人乳铁蛋白Human lactoferrin	山羊Capra hircus	3 200	2015	[6]
人血清白蛋白HSA	奶牛Bovine	2 500	2016	[7]
抗胰蛋白酶AAT	绵羊Ovis aries	650	2000	[8]
重组人纤溶酶原激活物 Recombinant human plasminogen activator	兔Leporidae	15.2-630	2016	[9]
凝血因子VIII FVlll	小鼠Mus musculus	1 000-4 000	2015	[10]
凝血因子IX FIX	猪Sus	100-400	2008	[11]
促红细胞生成素EPO	山羊Capra hircus	未报道Not reported	2002	[12]
人C1抑制剂Human C1 inhibitor	兔Leporidae	未报道Not reported	2003	[13]
人酸性α-葡萄糖苷酶 Human acid alpha-glucosidase	小鼠Mus musculus	1.5	1996	[14]
粒细胞巨噬细胞集落刺激因子GM-CSF	小鼠Mus musculus	200-4 600	1997	[15]
重组人抗凝血酶Recombinant human antithrombin	山羊Capra hircus	未报道Not reported	1998	[16]
人尿激酶Human urokinase	小鼠Mus musculus	1 000-2 000	1990	[17]
胰岛素样生长因子-1 IGF-1	兔Leporidae	1 000	1992	[18]
铜/锌超氧化物歧化酶、细胞外超氧化物歧化酶 CuZn-SOD、EC-SOD	山羊Capra hircus	100.14 ± 5.09、279.10 ± 5.38	2018	[19]
卵泡刺激素FSHα、FSHβ	山羊Capra hircus	0.28、0.30	2021	[20]
芳基烷基胺N-乙酰转移酶、乙酰5-羟色胺甲基转移酶AANAT、ASMT	绵羊Ovis aries	未报道Not reported	2017	[21]
葡糖脑苷脂酶Glucocerebrosidase	山羊Capra hircus	111.1 ± 8.1	2015	[22]
人蛋白C Human protein C	猪Sus	100-1 000	1994	[23]
抗程序性细胞死亡1抗体PD-1 antibody	小鼠Mus musculus	80.52 ± 0.82	2020	[24]
丁酰胆碱酯酶Butyrylcholinesterase	山羊Capra hircus	1 000-5 000	2008	[25]
生长激素Growth hormone	兔Leporidae	10	2012	[26]
胶原Collagen	小鼠Mus musculus	200	2000	[27]
抗CD20单克隆抗体Anti-CD20 mAB	小鼠Mus musculus	17	2008	[28]

打靶位点 Target site	物种 Species	转基因 Transgenes	基因编辑技术 Gene editing technology	时间/年 Time/Year	参考文献 Referencet
Rosa26	绵羊Ovis aries	turboGFP	CRISPR/Cas9	2016	[42]
	猪Sus	tdTomato	TALENs	2014	[43]
	牛Bovine	自然抗性相关巨噬细胞蛋白1 NRAMP1	CRISPR/Cas9	2021	[44]
β-casein	牛Bovine	人成纤维细胞生长因2 Human fibroblast growth factor 2	CRISPR/Cas9	2015	[45]
	绵羊Ovis aries	芳基烷基胺N-乙酰转移酶、乙酰5-羟色胺甲基转移酶AANAT、ASMT	CRISPR/Cas9	2017	[21]
	奶牛Bovine	溶葡球菌酶Lysostaphin	ZFNickases	2013	[46]
BLG	奶牛Bovine	人血清白蛋白HSA	TALENs	2016	[7]
	山羊Capra hircus	人α-乳清蛋白Human α -whey protein	Homologous recombination	2019	[41]
	山羊Capra hircus	人乳铁蛋白Human lactoferrin	CRISPR/Cas9	2017	[47]
	山羊Capra hircus	人乳铁蛋白Human lactoferrin	CRISPR/Cas9	2020	[39]
Col1a1	绵羊Ovis aries	抗胰蛋白酶AAT	Homologous recombination	2000	[8]
AAVS1	山羊Capra hircus	转化生长因子-β1 TGF-beta1	TALENs	2014	[48]
H11	猪Sus	绿色荧光蛋白GFP	CRISPR/Cas9	2015	[49]

打靶位点 Target site	物种 Species	转基因 Transgenes	基因编辑技术 Gene editing technology	时间/年 Time/Year	参考文献 Referencet
Rosa26	绵羊Ovis aries	turboGFP	CRISPR/Cas9	2016	[42]
	猪Sus	tdTomato	TALENs	2014	[43]
	牛Bovine	自然抗性相关巨噬细胞蛋白1 NRAMP1	CRISPR/Cas9	2021	[44]
β-casein	牛Bovine	人成纤维细胞生长因2 Human fibroblast growth factor 2	CRISPR/Cas9	2015	[45]
	绵羊Ovis aries	芳基烷基胺N-乙酰转移酶、乙酰5-羟色胺甲基转移酶AANAT、ASMT	CRISPR/Cas9	2017	[21]
	奶牛Bovine	溶葡球菌酶Lysostaphin	ZFNickases	2013	[46]
BLG	奶牛Bovine	人血清白蛋白HSA	TALENs	2016	[7]
	山羊Capra hircus	人α-乳清蛋白Human α -whey protein	Homologous recombination	2019	[41]
	山羊Capra hircus	人乳铁蛋白Human lactoferrin	CRISPR/Cas9	2017	[47]
	山羊Capra hircus	人乳铁蛋白Human lactoferrin	CRISPR/Cas9	2020	[39]
Col1a1	绵羊Ovis aries	抗胰蛋白酶AAT	Homologous recombination	2000	[8]
AAVS1	山羊Capra hircus	转化生长因子-β1 TGF-beta1	TALENs	2014	[48]
H11	猪Sus	绿色荧光蛋白GFP	CRISPR/Cas9	2015	[49]

类型 Type	名称 Name	转基因 Transgene	表达系统 Expression system	参考文献 Reference
启动子 Promoter	β-lactoglobulin promoter	溶菌酶 Lysozyme	山羊乳腺生物反应器 Goat mammary gland bioreactor	[75]
	αS1-casein promoter	抗程序性细胞死亡1抗体 PD-1 antibody	小鼠乳腺生物反应器 Mouse mammary gland bioreactor	[24]
	CMV promoter	凝血因子Ⅷ FVlll	小鼠乳腺生物反应器 Mouse mammary gland bioreactor	[10]
增强子 Enhancer	CMV enhancer	人乳铁蛋白 Human lactoferrin	山羊乳腺生物反应器 Goat mammary gland bioreactor	[76]
增强子 Enhancer	CMV enhancer	人乳铁蛋白 Human lactoferrin	小鼠乳腺生物反应器 Mouse mammary gland bioreactor	[77]
内含子 Intron	β-casein intron	溶菌酶 Lysozyme	小鼠乳腺生物反应器 Mouse mammary gland bioreactor	[78]
内含子 Intron	SV40 intron	促红细胞生成素 EPO	中国仓鼠卵巢细胞 CHO cell	[79]
绝缘子 Insulator	β-lactoglobulin insulator	溶菌酶 Lysozyme	猪乳腺生物反应器 Porcine mammary gland bioreactor	[80]
	cHS4 insulator	人血清白蛋白 HSA	牛乳腺生物反应器 Cattle mammary gland bioreactor	[81]
	tDNA insulator	单克隆抗体 Monoclonal antibody	中国仓鼠卵巢细胞 CHO cell	[82]
MAR	MAR X-29	增强型绿色荧光蛋白 eGFP	中国仓鼠卵巢细胞 CHO cell	[83]
	Chicken lysozyme MAR	免疫球蛋白 G IgG	中国仓鼠卵巢细胞 CHO cell	[84]
	IFN-β MAR	促红细胞生成素、肝细胞生长因子 EPO，HGF	中国仓鼠卵巢细胞 CHO cell	[85]
	β-globin MAR	可溶性TGF-II型受体 sTbetaRII	中国仓鼠卵巢细胞 CHO cell	[86]
	TOP1 MAR	增强型绿色荧光蛋白 eGFP	中国仓鼠卵巢细胞 CHO cell	[87]
	DHFR intron MAR	增强型绿色荧光蛋白 eGFP	中国仓鼠卵巢细胞 CHO cell	[88]
	MAR6	增强型绿色荧光蛋白 eGFP	中国仓鼠卵巢细胞 CHO cell	[88]
UCOE	UCOE	绿色荧光蛋白 GFP	中国仓鼠卵巢细胞 CHO cell	[89]
STAR	STAR7	单克隆抗体 Monoclonal antibody	中国仓鼠卵巢细胞 CHO cell	[90]
	STAR67	单克隆抗体 Monoclonal antibody	中国仓鼠卵巢细胞 CHO cell	[90]
	STAR40	分泌碱性磷酸酶 SEAP	中国仓鼠卵巢细胞 CHO cell	[91]