title: Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.
abstract: Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.
authors: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
authors: Eric J
authors: EJ
authors: Horstick
authors: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
authors: Diana C
authors: DC
authors: Jordan
authors: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
authors: Sadie A
authors: SA
authors: Bergeron
authors: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
authors: Kathryn M
authors: KM
authors: Tabor
authors: Program in Cellular Regulation and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
authors: Mihaela
authors: M
authors: Serpe
authors: Zebrafish Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
authors: Benjamin
authors: B
authors: Feldman
authors: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA haroldburgess@mail.nih.gov.
authors: Harold A
authors: http://orcid.org/0000-0003-1966-7801
authors: HA
authors: Burgess
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D003062:Codon; D019295:Computational Biology; D020869:Gene Expression Profiling; D008845:Microinjections; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D003062:Codon; D029961:Zebrafish Proteins
references: Science. 2013 Oct 25;342(6157):475-9
references: 24072823
references: Science. 2008 Jun 20;320(5883):1643-7
references: 18566288
references: Nucleic Acids Res. 1987 Feb 11;15(3):1281-95
references: 3547335
references: Gene Ther. 2009 May;16(5):605-19
references: 19262615
references: Nucleic Acids Res. 2000 Jan 1;28(1):292
references: 10592250
references: PLoS One. 2008;3(11):e3621
references: 18978947
references: RNA. 2003 May;9(5):607-17
references: 12702819
references: Mar Biotechnol (NY). 2000 Mar;2(2):107-125
references: 10811950
references: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13365-70
references: 19628697
references: Science. 2013 Dec 13;342(6164):1367-72
references: 24337295
references: Nucleic Acids Res. 1990 Nov 11;18(21):6339-45
references: 2123028
references: Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2122-7
references: 9122158
references: Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1255-60
references: 18202183
references: J Neurosci. 2012 Oct 24;32(43):15205-15
references: 23100441
references: J Mol Biol. 1996 Oct 4;262(4):459-72
references: 8893856
references: Gene. 1991 Dec 15;108(2):193-9
references: 1660837
references: Bioinformation. 2009 Sep 05;4(2):63-5
references: 20198170
references: Genetics. 2006 Oct;174(2):639-49
references: 16959904
references: PLoS Comput Biol. 2012;8(11):e1002755
references: 23133360
references: Methods. 2014 Apr 1;66(3):433-40
references: 23792917
references: Nucleic Acids Res. 1990 Mar 25;18(6):1465-73
references: 2109307
references: Development. 1988 Jun;103(2):403-12
references: 2852096
references: Science. 2009 Apr 10;324(5924):255-8
references: 19359587
references: Gene. 2011 Nov 15;488(1-2):35-40
references: 21914464
references: Development. 2010 Jun;137(12):1981-90
references: 20463035
references: Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):E4316-24
references: 24151332
references: Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1027-32
references: 17209010
references: Genetics. 1998 May;149(1):37-44
references: 9584084
references: EMBO J. 2002 Aug 15;21(16):4357-67
references: 12169638
references: Front Neural Circuits. 2012 Dec 28;6:110
references: 23293587
references: EMBO Rep. 2001 Sep;2(9):787-93
references: 11520858
references: Nucleic Acids Res. 1997 Mar 1;25(5):955-64
references: 9023104
references: Genome Biol Evol. 2011;3:1187-96
references: 21920901
references: Cell. 1986 Jan 31;44(2):283-92
references: 3943125
references: Methods Cell Biol. 2004;77:201-22
references: 15602913
references: Science. 2006 Dec 22;314(5807):1930-3
references: 17185601
references: Dev Cell. 2004 Jan;6(1):117-31
references: 14723852
references: RNA. 2012 Jul;18(7):1358-72
references: 22586155
references: PLoS One. 2007;2(2):e188
references: 17285142
references: PLoS One. 2008;3(10):e3412
references: 18923675
references: Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1358-62
references: 1741388
references: Genome Res. 2004 Jun;14(6):1188-90
references: 15173120
references: Mech Dev. 2007 Mar;124(3):218-29
references: 17223324
references: Mol Mar Biol Biotechnol. 1992 Aug-Oct;1(4-5):290-300
references: 1308820
references: Genome Res. 2010 Jun;20(6):804-15
references: 20219939
references: J Biotechnol. 2007 Aug 1;131(1):1-8
references: 17617485
references: Cell Mol Life Sci. 2012 Nov;69(21):3613-34
references: 22538991
references: Proteomics. 2008 Mar;8(6):1257-65
references: 18283662
references: Transgenic Res. 2010 Apr;19(2):299-304
references: 19662507
references: Cell. 2010 Apr 30;141(3):536-48
references: 20434990
references: Biotechniques. 2000 Sep;29(3):636-42
references: 10997278
references: Nucleic Acids Res. 1984 Sep 25;12(18):7057-70
references: 6207484
references: Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14338-43
references: 16176987
references: Mol Psychiatry. 2015 Aug;20(8):974-85
references: 25224259
references: Plant J. 2004 Dec;40(5):744-51
references: 15546357
references: Protein Expr Purif. 2008 May;59(1):94-102
references: 18289875
references: Methods Mol Biol. 2008;453:3-31
references: 18712296
references: EMBO J. 1990 Nov;9(11):3743-51
references: 2145153
references: J Mol Biol. 1966 Aug;19(2):548-55
references: 5969078
references: Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11811-5
references: 8265631
references: New Biol. 1990 May;2(5):441-9
references: 2288914
references: Nat Genet. 2002 Aug;31(4):415-8
references: 12134150
references: PLoS Comput Biol. 2011 Sep;7(9):e1002150
references: 21935348
references: Dev Dyn. 2009 Mar;238(3):641-55
references: 19191223
references: Cell. 2010 Apr 16;141(2):344-54
references: 20403328
references: J Mol Biol. 2007 Feb 9;366(1):1-13
references: 17187822
references: Science. 2007 Jan 26;315(5811):525-8
references: 17185560
references: Development. 2008 Sep;135(18):3001-5
references: 18684740
references: Trends Biochem Sci. 1995 Nov;20(11):465-70
references: 8578590
references: Cell. 2010 Apr 16;141(2):355-67
references: 20403329
references: Genetics. 2009 Jul;182(3):747-55
references: 19433629
references: Nucleic Acids Res. 2006;34(7):1959-73
references: 16614446
references: EMBO J. 1995 Jan 3;14(1):151-8
references: 7828587
references: Genome Res. 2003 Dec;13(12):2665-73
references: 14656971
references: PLoS One. 2013;8(6):e65932
references: 23776572
references: Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6626-31
references: 22493255
references: Dev Dyn. 2007 Apr;236(4):1025-35
references: 17326133
references: Nat Biotechnol. 2004 Apr;22(4):445-9
references: 14990965
references: Nat Methods. 2008 Jun;5(6):545-51
references: 18454154
references: BMC Genomics. 2009;10:274
references: 19538749
references: Genetics. 2010 Oct;186(2):735-55
references: 20697123
references: Mol Cell Biol. 1991 Jun;11(6):3070-4
references: 2038318
references: J Neurophysiol. 2014 Aug 15;112(4):834-44
references: 24848468
references: Genes Dev. 1999 Mar 15;13(6):633-6
references: 10090718
references: Gene Ther. 2006 Apr;13(7):641-5
references: 16355114
references: Genes Dev. 2004 Jan 15;18(2):210-22
references: 14752011
references: PLoS One. 2013;8(12):e85218
references: 24391998
references: Pathology. 2007 Apr;39(2):247-51
references: 17454756
references: Dev Dyn. 2007 Nov;236(11):3088-99
references: 17937395
references: Dev Dyn. 2008 Sep;237(9):2466-74
references: 18729212