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W1967785063 abstract "Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA[Ser]Sec gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T→A) or 37 (37A→G) in tRNA[Ser]Sec. The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2′-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N6-isopentenyladenosine. Both mutant tRNAs lacked the 2′-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with 75Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins. Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA[Ser]Sec gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T→A) or 37 (37A→G) in tRNA[Ser]Sec. The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2′-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N6-isopentenyladenosine. Both mutant tRNAs lacked the 2′-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with 75Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins. There are 24 known selenoproteins in rodents and 25 in humans (1Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1839) Google Scholar). The targeted removal of specific selenoproteins has shown that some are essential in development, whereas others appear to be nonessential. For example, the loss of selenoproteins glutathione peroxidase 4 (GPx4) (2Yant L.J. Ran Q. Rao L. Remmen VanH. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (541) Google Scholar) or thioredoxin reductase 1 (TR1 or Txnrd1) (3Jakupoglu C. Przemeck G.K. Schneider M. Moreno S.G. Mayr N. Hatzopoulos A.K. de Angelis M.H. Wurst W. Bornkamm G.W. Brielmeier M. Conrad M. Mol. Cell. Biol. 2005; 25: 1980-1988Crossref PubMed Scopus (289) Google Scholar) or 2 (TR3 or Txnrd2) (4Conrad M. Jakupoglu C. Moreno S.G. Lippl S. Banjac A. Schneider M. Beck H. Hatzopoulos A.K. Just U. Sinowatz F. Schmahl W. Chien K.R. Wurst W. Bornkamm G.W. Brielmeier M. Mol. Cell. Biol. 2004; 24: 9414-9423Crossref PubMed Scopus (390) Google Scholar) is embryonic lethal, whereas the loss of glutathione peroxidase 1 (GPx1) (5Ho Y.S. Magnenat J.L. Bronson R.T. Cao J. Gargano M. Sugawara M. Funk C.D. J. Biol. Chem. 1997; 272: 16644-16651Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar) or 2 (GPx2) (6Esworthy R.S. Aranda R. Martin M.G. Doroshow J.H. Binder S.W. Chu F.F. Am. J. Physiol. 2001; 281: G848-G855Crossref PubMed Google Scholar) appears to be of little or no consequence. Other studies, however, suggest that those selenoproteins whose loss results in little or no phenotypic change may function in protective mechanisms against certain environmental stresses (see Ref. 6Esworthy R.S. Aranda R. Martin M.G. Doroshow J.H. Binder S.W. Chu F.F. Am. J. Physiol. 2001; 281: G848-G855Crossref PubMed Google Scholar and references therein). There are selenoproteins whose removal or mutation results in dramatic effects on health. For example, knock-out of selenoprotein P (SelP) 6The abbreviations used are: SelP, selenoprotein P; GPx, glutathione peroxidase; TR, thioredoxin reductase; Sec, selenocysteine; mcm5U, 5-methoxycarbonylmethyluridine; mcm5Um, 5-methoxycarbonylmethyl-2′-O-methyluridine; i6A, isopentenyladenosine; NMD, nonsense-mediated decay. 6The abbreviations used are: SelP, selenoprotein P; GPx, glutathione peroxidase; TR, thioredoxin reductase; Sec, selenocysteine; mcm5U, 5-methoxycarbonylmethyluridine; mcm5Um, 5-methoxycarbonylmethyl-2′-O-methyluridine; i6A, isopentenyladenosine; NMD, nonsense-mediated decay. causes neurological problems (7Hill K.E. Zhou J. McMahan W.J. Motley A.K. Atkins J.F. Gesteland R.F. Burk R.F. J. Biol. Chem. 2003; 278: 13640-13646Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 8Schomburg L. Schweizer U. Holtmann B. Flohe L. Sendtner M. Kohrle J. Biochem. J. 2003; 370: 397-402Crossref PubMed Scopus (349) Google Scholar), and knock-out of type 2 iodothyronine deiodinase results in a variety of defects, including an impaired adaptive thermogenesis and hypothermia in cold-exposed mice (see Ref. 9Christoffolete M.A. Linardi C.C. de Jesus L. Ebina K.N. Carvalho S.D. Ribeiro M.O. Rabelo R. Curcio C. Martins L. Kimura E.T. Bianco A.C. Diabetes. 2004; 53: 577-584Crossref PubMed Scopus (171) Google Scholar and references therein), retarded cochlear development and hearing loss (10Ng L. Goodyear R.J. Woods C.A. Schneider M.J. Diamond E. Richardson G.P. Kelley M.W. Germain D.L. Galton V.A. Forrest D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3474-3479Crossref PubMed Scopus (194) Google Scholar), and a pituitary resistance to thyroxine (11de Jesus L.A. Carvalho S.D. Ribeiro M.O. Schneider M. Kim S.W. Harney J.W. Larsen P.R. Bianco A.C. J. Clin. Investig. 2001; 108: 1379-1385Crossref PubMed Scopus (398) Google Scholar). Mutations affecting selenoprotein N (SelN result in several muscle disorders (12Petit N. Lescure A. Rederstorff M. Krol A. Moghadaszadeh B. Wewer U.M. Guicheney P. Hum. Mol. Genet. 2003; 12: 1045-1053Crossref PubMed Scopus (153) Google Scholar, 13Tajsharghi H. Darin N. Tulinius M. Oldfors A. Neuromuscul. Disord. 2005; 15: 299-302Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). LoxP-Cre technology, which allows the removal of embryonic lethal genes in specific tissues and organs (3Jakupoglu C. Przemeck G.K. Schneider M. Moreno S.G. Mayr N. Hatzopoulos A.K. de Angelis M.H. Wurst W. Bornkamm G.W. Brielmeier M. Conrad M. Mol. Cell. Biol. 2005; 25: 1980-1988Crossref PubMed Scopus (289) Google Scholar, 4Conrad M. Jakupoglu C. Moreno S.G. Lippl S. Banjac A. Schneider M. Beck H. Hatzopoulos A.K. Just U. Sinowatz F. Schmahl W. Chien K.R. Wurst W. Bornkamm G.W. Brielmeier M. Mol. Cell. Biol. 2004; 24: 9414-9423Crossref PubMed Scopus (390) Google Scholar, 14Conrad M. Moreno S.G. Sinowatz F. Ursini F. Kolle S. Roveri A. Brielmeier M. Wurst W. Maiorino M. Bornkamm G.W. Mol. Cell. Biol. 2005; 25: 7637-7644Crossref PubMed Scopus (203) Google Scholar), has been used to examine the roles of essential selenoprotein genes in development and health. Such studies have elucidated key roles of TR1 in embryogenesis of numerous tissues and organs, except heart (3Jakupoglu C. Przemeck G.K. Schneider M. Moreno S.G. Mayr N. Hatzopoulos A.K. de Angelis M.H. Wurst W. Bornkamm G.W. Brielmeier M. Conrad M. Mol. Cell. Biol. 2005; 25: 1980-1988Crossref PubMed Scopus (289) Google Scholar), and of TR3 in hematopoiesis and in heart development and function (4Conrad M. Jakupoglu C. Moreno S.G. Lippl S. Banjac A. Schneider M. Beck H. Hatzopoulos A.K. Just U. Sinowatz F. Schmahl W. Chien K.R. Wurst W. Bornkamm G.W. Brielmeier M. Mol. Cell. Biol. 2004; 24: 9414-9423Crossref PubMed Scopus (390) Google Scholar). The targeted removal of the nuclear form of GPx4 (designated snGPx4) results in viable and completely fertile animals, although the overall structural stability of sperm chromatin is diminished (14Conrad M. Moreno S.G. Sinowatz F. Ursini F. Kolle S. Roveri A. Brielmeier M. Wurst W. Maiorino M. Bornkamm G.W. Mol. Cell. Biol. 2005; 25: 7637-7644Crossref PubMed Scopus (203) Google Scholar). Loss of SelP in liver, achieved by targeted knock-out of the selenocysteine (Sec) tRNA[Ser]Sec gene (designated trsp), implicated SelP in transport functions in plasma and substantiated its essential role in brain (15Schweizer U. Streckfuss F. Pelt P. Carlson B.A. Hatfield D.L. Kohrle J. Schomburg L. Biochem. J. 2005; 386: 221-226Crossref PubMed Scopus (172) Google Scholar). Selenoprotein synthesis is dependent on the presence of Sec tRNA[Ser]Sec. Given this dependence, selenoprotein expression can be modulated by perturbing Sec tRNA[Ser]Sec expression, providing a means of elucidating the role of selenoproteins and selenium in development and health (16Moustafa M.E. Kumaraswamy E. Zhong N. Rao M. Carlson B.A. Hatfield D.L. J. Nutr. 2003; 133: S2494-S2496Crossref Google Scholar). The Sec tRNA[Ser]Sec population in higher vertebrates consists of two isoforms that differ by a single 2′-O-methyl group. One isoform contains 5-methoxycarbonylmethyluridine (mcm5U) at position 34, and the other is methylated on the ribosyl moiety at that position generating 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um; see Ref. 17Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (543) Google Scholar). The presence of this 2′-methyl ribose modification (designated Um34) confers several unique properties on mcm5Um. For example, Um34 affects Sec tRNA[Ser]SecmcmUm secondary and tertiary structure (18Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C.J. Hill K.E. Burk R.F. McCloskey J.A. Hatfield D.L. J. Biol. Chem. 1993; 268: 14215-14223Abstract Full Text PDF PubMed Google Scholar). Um34 addition is dependent on the prior synthesis of the four modified bases found in tRNA[Ser]Sec and on an intact tertiary structure (19Kim L.K. Matsufuji T. Matsufuji S. Carlson B.A. Kim S.S. Hatfield D.L. Lee B.J. RNA (N. Y.). 2000; 6: 1306-1315Crossref PubMed Scopus (55) Google Scholar). Synthesis of all other modified nucleosides of Sec tRNA[Ser]Sec, including mcm5Um, is less stringently associated with primary and tertiary structure. In addition, synthesis of Um34 is dependent on the selenium status of the organism, with increased dietary selenium increasing Um34 levels (17Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (543) Google Scholar). Removal of trsp is embryonic lethal (20Bösl M.R. Takaku K. Oshima M. Nishimura S. Taketo M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5531-5534Crossref PubMed Scopus (281) Google Scholar, 21Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (93) Google Scholar). Therefore, to alter the Sec tRNA[Ser]Sec population, techniques of influencing Sec tRNA[Ser]Sec levels other than the sole removal of trsp must be employed. We previously generated transgenic mice with extra copies of wild type or mutant Sec tRNA[Ser]Sec transgenes (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar) and mice with a conditional knock-out of trsp (21Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (93) Google Scholar), and we then rescued selenoprotein expression in trsp null mice with wild type or mutant Sec tRNA[Ser]Sec transgenes (23Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2005; 280: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. Topics in Current Genetics. 2005; (Grosjean, H., ed) pp. , Springer, Berlin-Heidelberg, Germany: 431-438Crossref Google Scholar). Consistent with reports that the Sec tRNA[Ser]Sec population is not limiting in selenoprotein biosynthesis (20Bösl M.R. Takaku K. Oshima M. Nishimura S. Taketo M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5531-5534Crossref PubMed Scopus (281) Google Scholar, 22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar, 25Chittum H.S. Hill K.E. Carlson B.A. Lee B.J. Burk R.F. Hatfield D.L. Biochim. Biophys. Acta. 1997; 1359: 25-34Crossref PubMed Scopus (48) Google Scholar), we found little or no effect of extra copies of wild type transgenes on selenoprotein expression in the tissues and cells examined (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar). In contrast, multiple copies of a mutant trsp transgene can lead to specific altercations in the selenoprotein population (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar). For example, transgenes with a mutation at position 37 (37A→G) produce a tRNA gene product that not only lacks isopentenyladenosine (i6A) at this site but also lacks Um34 (19Kim L.K. Matsufuji T. Matsufuji S. Carlson B.A. Kim S.S. Hatfield D.L. Lee B.J. RNA (N. Y.). 2000; 6: 1306-1315Crossref PubMed Scopus (55) Google Scholar). Selenoprotein synthesis was affected in mice carrying the G37 Sec tRNA[Ser]Sec transgene in a protein- and tissue-specific manner (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar). Rescue of selenoprotein expression in trsp null mice with the G37 Sec tRNA[Ser]Sec transgene results in the recovery of housekeeping selenoproteins, whereas numerous stress-related selenoproteins that are nonessential to survival are either not rescued or are poorly rescued (23Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2005; 280: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. Topics in Current Genetics. 2005; (Grosjean, H., ed) pp. , Springer, Berlin-Heidelberg, Germany: 431-438Crossref Google Scholar). Although the wild type and mutant trsp transgenic models and transgenic trsp rescue models have provided considerable insight into selenoprotein expression and the hierarchy of selenoprotein expression (21Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (93) Google Scholar, 22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar, 23Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2005; 280: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. Topics in Current Genetics. 2005; (Grosjean, H., ed) pp. , Springer, Berlin-Heidelberg, Germany: 431-438Crossref Google Scholar), they have limitations. For example, when expressing mutant trsp transgenes in mice carrying the endogenous allele of trsp, expression from the wild type trsp can confound the studies. Studies with rescue models like that described above, with a germ line conditional trsp allele, focus on the selenoprotein population in the whole animal. The targeted removal of floxed trsp in defined cell types using transgenic mice with tissue-specific expression of Cre recombinase permitted some study of the effects of selenoprotein loss in specific tissues and organs in the absence of endogenous trsp. However, the resulting animals have a variety of defects, including embryonic mortality or early adult death (26Shrimali R.K. Weaver J.A. Miller G.F. Carlson B.A. Novoselov S.N. Kumaraswamy E. Gladyshev V.N. Hatfield D.L. Neuromuscul. Disord. 2007; 17: 135-142Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), thus restricting the use of these models for studying the role of selenium and selenoproteins in health. In this study, we generated a mouse model that targets the removal of trsp in liver for use in elucidating the role of selenium and selenoproteins and the contributions of housekeeping and stress-related selenoproteins in health. trsp loxP-albumin Cre mice (27Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) were crossed with i6A-Um34-deficient Sec tRNA[Ser]Sec transgenic mice (designated here as G37 transgenic mice (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar)) or with another mutant trsp (34T→A) transgenic mouse described here that lacks mcm5U and consequently the Um34 modification (designated here as A34 transgenic mice). The resulting mouse lines lack trsp in liver and are dependent on the A34 mutant transgene or the G37 mutant transgene for selenoprotein expression. These new mouse models provide us with novel experimental systems for investigating the role of numerous stress-related selenoproteins in health in a specifically targeted organ. Materials—[75Se]Selenium (specific activity 1000 Ci/mmol) was obtained from the Research Reactor Facility, University of Missouri, Columbia; [3H]serine (specific activity 29 Ci/mmol) was from GE Healthcare, and [α-32P]dCTP (specific activity ∼6000 Ci/mmol) was from PerkinElmer Life Sciences. Hybond Nylon N+ membranes were purchased from GE Healthcare; NuPAGE 10% polyacrylamide gels, polyvinylidene difluoride membranes, Superscript II reverse transcriptase, and SeeBlue Plus2 protein markers were from Invitrogen; SuperSignal West Dura extended duration substrate was from Pierce; Universal Reference RNA was from Stratagene; ADP-Sepharose 4B resin was from GE Healthcare; and anti-rabbit horseradish peroxidase-conjugated and anti-chicken horseradish peroxidase-conjugated secondary antibodies were from Sigma. Reagents for the TR1 assay (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar) were purchased from Sigma. Antibodies against GPx1 were obtained from Abcam, and antibodies against GPx4, TR1, TR3, SelR, and SelT were from our laboratories (1Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1839) Google Scholar, 24Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. Topics in Current Genetics. 2005; (Grosjean, H., ed) pp. , Springer, Berlin-Heidelberg, Germany: 431-438Crossref Google Scholar, 27Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). All other reagents were obtained commercially and were products of the highest grade available. Animals and Genotyping of Mice—Homozygous floxed trsp C57BL/6 mice (trspfl) that were also homozygous for albumin Cre (AlbCre) were designated Δtrsp after trspfl was removed by the Cre recombinase (23Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2005; 280: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24Carlson B.A. Xu X.M. Gladyshev V.N. Hatfield D.L. Topics in Current Genetics. 2005; (Grosjean, H., ed) pp. , Springer, Berlin-Heidelberg, Germany: 431-438Crossref Google Scholar, 27Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Δtrsp C57BL/6-FVB/N transgenic mice were homozygous for one of three types of trsp transgene (trspt) alleles as follows: 1) wild type transgene encoding 10 copies of wild type trspt/allele (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar); 2) G37 low or high copy transgene encoding either 1 (low) or 8 (high) copies of the 37A→G mutant trspt/allele; or 3) A34 transgene encoding one copy of the 34T→A mutant trspt/allele. The product of the G37 transgene lacks the highly modified base, i6A, at position 37 and also Um34. The single copy G37 transgenic mouse was generated specifically for this study (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar) to compare with the effects of the single copy A34 transgene. The product of the A34 transgene lacked the highly modified base, mcm5U, and also lacked Um34. 34T→A transgenic mice were generated exactly as described (22Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar) except that the transgene construct contained an A at position 34 instead of a T, and the base at position 37 was the wild type A base; and three founders that were heterozygous for 1, 4, and 6 transgene copies were obtained. A34 transgenic mice were in strain FVB/N, and founders were bred to obtain the corresponding homozygous mice (designated A34-2, A34-8, and A34-12, respectively). Genotype designations and definitions are given in the legend of Table 1. All mice used in this study were males. Matings to obtain mouse lines carrying wild type, A34, and G37 transgenes and Δtrsp in their liver are summarized in Table 1.TABLE 1Summary of breeding schemes for generating experimental mouse linesMatingsaMatings to obtain each mouse line used in this study are shown in the table.Wild typebBoth wild type mice, designated trsp in the text, and wild type selenoprotein replacement mice, designated trspt in the text, were used as control mice.,cGenotype designations denote the following: trsp+ (wild type Sec tRNA[Ser]Sec gene), trspfl (floxed Sec tRNA[Ser]Sec gene); trspt (Sec tRNA[Ser]Sec transgene); Δtrsp (liver trsp knockout); AlbCre (albumin Cre gene); A34 (A34 mutant transgene); and G37 (G37 mutant transgene). Homozygous genotypes and the corresponding heterozygous genotypes are designated as follows: trsp+/+ and trsp+/- (wild type Sec tRNA[Ser]Sec gene); trspfl/fl and trspfl/+ (floxed Sec tRNA[Ser]Sec gene); trspt/t and trspt/- (wild type Sec tRNA[Ser]Sec transgene); AlbCre+/+ and AlbCre+/- (albumin Cre gene); A34t/t and A34t/- (A34 transgene); and G37t/t and G37t/- (G37 transgene).Wild type replacement,bBoth wild type mice, designated trsp in the text, and wild type selenoprotein replacement mice, designated trspt in the text, were used as control mice.,cGenotype designations denote the following: trsp+ (wild type Sec tRNA[Ser]Sec gene), trspfl (floxed Sec tRNA[Ser]Sec gene); trspt (Sec tRNA[Ser]Sec transgene); Δtrsp (liver trsp knockout); AlbCre (albumin Cre gene); A34 (A34 mutant transgene); and G37 (G37 mutant transgene). Homozygous genotypes and the corresponding heterozygous genotypes are designated as follows: trsp+/+ and trsp+/- (wild type Sec tRNA[Ser]Sec gene); trspfl/fl and trspfl/+ (floxed Sec tRNA[Ser]Sec gene); trspt/t and trspt/- (wild type Sec tRNA[Ser]Sec transgene); AlbCre+/+ and AlbCre+/- (albumin Cre gene); A34t/t and A34t/- (A34 transgene); and G37t/t and G37t/- (G37 transgene). (20 copies)A34 transgenic,cGenotype designations denote the following: trsp+ (wild type Sec tRNA[Ser]Sec gene), trspfl (floxed Sec tRNA[Ser]Sec gene); trspt (Sec tRNA[Ser]Sec transgene); Δtrsp (liver trsp knockout); AlbCre (albumin Cre gene); A34 (A34 mutant transgene); and G37 (G37 mutant transgene). Homozygous genotypes and the corresponding heterozygous genotypes are designated as follows: trsp+/+ and trsp+/- (wild type Sec tRNA[Ser]Sec gene); trspfl/fl and trspfl/+ (floxed Sec tRNA[Ser]Sec gene); trspt/t and trspt/- (wild type Sec tRNA[Ser]Sec transgene); AlbCre+/+ and AlbCre+/- (albumin Cre gene); A34t/t and A34t/- (A34 transgene); and G37t/t and G37t/- (G37 transgene). (2 copies)G37 transgenic,cGenotype designations denote the following: trsp+ (wild type Sec tRNA[Ser]Sec gene), trspfl (floxed Sec tRNA[Ser]Sec gene); trspt (Sec tRNA[Ser]Sec transgene); Δtrsp (liver trsp knockout); AlbCre (albumin Cre gene); A34 (A34 mutant transgene); and G37 (G37 mutant transgene). Homozygous genotypes and the corresponding heterozygous genotypes are designated as follows: trsp+/+ and trsp+/- (wild type Sec tRNA[Ser]Sec gene); trspfl/fl and trspfl/+ (floxed Sec tRNA[Ser]Sec gene); trspt/t and trspt/- (wild type Sec tRNA[Ser]Sec transgene); AlbCre+/+ and AlbCre+/- (albumin Cre gene); A34t/t and A34t/- (A34 transgene); and G37t/t and G37t/- (G37 transgene). (2 or 16 copies)dG37 transgenic mice carrying 2 or 16 copies of the transgene are referred to in the text as G37 low copy number or G37 high copy number, respectively.Δtrsp knockoutcGenotype designations denote the following: trsp+ (wild type Sec tRNA[Ser]Sec gene), trspfl (floxed Sec tRNA[Ser]Sec gene); trspt (Sec tRNA[Ser]Sec transgene); Δtrsp (liver trsp knockout); AlbCre (albumin Cre gene); A34 (A34 mutant transgene); and G37 (G37 mutant transgene). Homozygous genotypes and the corresponding heterozygous genotypes are designated as follows: trsp+/+ and trsp+/- (wild type Sec tRNA[Ser]Sec gene); trspfl/fl and trspfl/+ (floxed Sec tRNA[Ser]Sec gene); trspt/t and trspt/- (wild type Sec tRNA[Ser]Sec transgene); AlbCre+/+ and AlbCre+/- (albumin Cre gene); A34t/t and A34t/- (A34 transgene); and G37t/t and G37t/- (G37 transgene).1st breedingtrsp+/+ × trspfl/+-AlbCre+/+trspt/t × trspfl/+-AlbCre+/+trsp+/+-A34t/t × trspfl/+-AlbCre+/+trsp+/+-G37t/t × trspfl/+-AlbCre+/+trspfl/f × trspfl/+-AlbCre+/+F1 (2nd breeding)trsp+/+-AlbCre+/- × trsp+/+-AlbCre+/-trspfl/+-AlbCre+/--trspt/- × trspfl/+-AlbCre+/--trspt/-trspfl/+-AlbCre+/--A34tt/- × trspfl/+-AlbCre+/--A34t/-trspfl/+-AlbCre+/--G37tt/- × trspfl/+-AlbCre+/--G37t/-trspfl/+-AlbCre+/- × trspfl/+-AlbCre+/-F2 (experimental mice)eExperimental mice are those animals generated from the matings for use in the study.trsp+/+-AlbCre+/+trspfl/fl-AlbCre+/+-trspt/ttrspfl/fl-AlbCre+/+-A34t/ttrspfl/fl-AlbCre+/+-G37t/ttrspfl/fl-AlbCre+/+DesignationfThe genotypes shown in the F2 experimental mice are designated in the text as follows: trsp or wild type control mouse; trspt or wild type replacement mouse, which is also a control mouse; A34 transgenic mouse; G37 transgenic mouse; Δtrsp or liver knock-out mouse.trsptrsptA34G37Δtrspa Matings to obtain each mouse line used in this study are shown in the table.b Both wild type mice, designated trsp in the text, and wild type selenoprotein replacement mice, designated trspt in the text, were used as control mice.c Genotype designations denote the following: trsp+ (wild type Sec tRNA" @default.
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W1967785063 title "Selective Restoration of the Selenoprotein Population in a Mouse Hepatocyte Selenoproteinless Background with Different Mutant Selenocysteine tRNAs Lacking Um34" @default.
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