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• W2092003249 abstract "Consistent expression from CD45 cDNA constructs has proven difficult to achieve. Through the use of new CD45 cDNA constructs and reporter genes, the role 5′, 3′, and intron sequences play in CD45 expression was determined. The CD45 polyadenylation signal sequence was fully functional in a β-galactosidase reporter construct. Furthermore, the CD45 3′-untranslated region and downstream sequences were shown to contain no negative regulatory elements. Several new CD45 cDNA constructs were designed that contain either the cytomegalovirus promoter, the leukocyte function-associated antigen (LFA-1; CD11a) promoter, or various CD45 5′ regions. Neither the cytomegalovirus nor the LFA-1 promoter was capable of generating detectable levels of expression in constructs with CD45 cDNA. However, when CD45 intron sequences between exons 3 and 9 were inserted in the cDNA construct to generate a CD45 minigene, the LFA-1 promoter was able to drive reproducible, significant expression of CD45. CD45 minigenes using the CD45 5′ sequences up to 19 kilobases upstream of the transcriptional start produced very little protein. The LFA-1 CD45 minigene construct produced correct cell type-specific isoforms when expressed in T and B lymphocyte lines. Therefore, we conclude that the regulation of CD45 expression and cell type-specific splicing requires elements within the intron sequences. Consistent expression from CD45 cDNA constructs has proven difficult to achieve. Through the use of new CD45 cDNA constructs and reporter genes, the role 5′, 3′, and intron sequences play in CD45 expression was determined. The CD45 polyadenylation signal sequence was fully functional in a β-galactosidase reporter construct. Furthermore, the CD45 3′-untranslated region and downstream sequences were shown to contain no negative regulatory elements. Several new CD45 cDNA constructs were designed that contain either the cytomegalovirus promoter, the leukocyte function-associated antigen (LFA-1; CD11a) promoter, or various CD45 5′ regions. Neither the cytomegalovirus nor the LFA-1 promoter was capable of generating detectable levels of expression in constructs with CD45 cDNA. However, when CD45 intron sequences between exons 3 and 9 were inserted in the cDNA construct to generate a CD45 minigene, the LFA-1 promoter was able to drive reproducible, significant expression of CD45. CD45 minigenes using the CD45 5′ sequences up to 19 kilobases upstream of the transcriptional start produced very little protein. The LFA-1 CD45 minigene construct produced correct cell type-specific isoforms when expressed in T and B lymphocyte lines. Therefore, we conclude that the regulation of CD45 expression and cell type-specific splicing requires elements within the intron sequences. kilobase(s) base pair(s) phosphate-buffered saline polymerase chain reaction reverse transcription cytomegalovirus fluorescein isothiocyanate leukocyte function-associated antigen untranslated region CD45 is a high molecular weight, transmembrane protein-tyrosine phosphatase expressed on all nucleated cells of hematopoietic origin. The protein is structurally heterogenous, consisting of isoforms ranging in size from 180 to 220 kDa. The heterogeneity of the protein results from differential RNA splicing of at least five and perhaps six exons encoding part of the extracellular domain (1Chang H.L. Lefrancois L. Zaroukian M.H. Esselman W.J. J. Immunol. 1991; 147: 1687-1693PubMed Google Scholar, 2Virts E. Barritt D. Raschke W.C. Mol. Immunol. 1998; 35: 167-176Crossref PubMed Scopus (18) Google Scholar, 3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar). The CD45 exon usage pattern is highly regulated by the different leukocyte populations and is cell type-specific. The predominant isoform expressed by B cells contains all variable exons (B220+) (4Shen F.W. Saga Y. Litman G. Freeman G. Tung J.S. Cantor H. Boyse E.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7360-7363Crossref PubMed Scopus (82) Google Scholar, 5Thomas M.L. Reynolds P.J. Chain A. Ben-Neriah Y. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5360-5363Crossref PubMed Scopus (84) Google Scholar). Upon antigen stimulation, lower molecular weight isoforms are detected (6Hathcock K.S. Hirano H. Murakami S. Hodes R.J. J. Immunol. 1992; 149: 2286-2294PubMed Google Scholar, 7Ogimoto M. Katagiri T. Hasegawa K. Mizuno K. Yakura H. Cell. Immunol. 1993; 151: 97-109Crossref PubMed Scopus (15) Google Scholar). Several subtypes of antigen-specific memory B cells have been identified, including an antibody-secreting subset producing the full-length splice variant and a nonsecreting subtype, which is B220− (8McHeyzer-Williams L.J. Cool M. McHeyzer-Williams M.G. J. Exp. Med. 2000; 191: 1149-1166Crossref PubMed Scopus (142) Google Scholar). In thymocytes three or four variable exons are removed to generate the lowest molecular weight isoforms (1Chang H.L. Lefrancois L. Zaroukian M.H. Esselman W.J. J. Immunol. 1991; 147: 1687-1693PubMed Google Scholar, 3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar, 4Shen F.W. Saga Y. Litman G. Freeman G. Tung J.S. Cantor H. Boyse E.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7360-7363Crossref PubMed Scopus (82) Google Scholar,9Chang H.L. Zaroukian M.H. Esselman W.J. J. Immunol. 1989; 143: 315-321PubMed Google Scholar, 10Saga Y. Tung J.S. Shen F.W. Boyse E.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5364-5368Crossref PubMed Scopus (69) Google Scholar, 11Saga Y. Furukawa K. Rogers P. Tung J.S. Parker D. Boyse E.A. Immunogenetics. 1990; 31: 296-306Crossref PubMed Scopus (15) Google Scholar). Peripheral T cells express multiple splice variants and have a varied isoform expression pattern that is dependent upon the differentiation state, function, and prior antigenic exposure (2Virts E. Barritt D. Raschke W.C. Mol. Immunol. 1998; 35: 167-176Crossref PubMed Scopus (18) Google Scholar). Mast cells and monocytes also produce specific sets of CD45 isoforms, which are distinctive for each myleoid cell type (12Virts E. Barritt D. Siden E. Raschke W.C. Mol. Immunol. 1997; 34: 1191-1197Crossref PubMed Scopus (7) Google Scholar). In addition to the protein structural differences, polymorphic sequence variations in the CD45 gene led to the identification of three alleles in inbred murine strains, Ly5 a (CD45.1),Ly5 b (CD45.2), and Ly5 c (13Seldin M.F. D'Hoostelaere L.A. Steinberg A.D. Saga Y. Morse H.C.D. Immunogenetics. 1987; 26: 74-78Crossref PubMed Scopus (15) Google Scholar). The CD45.2 allele is expressed by most of the established strains, while the CD45.1 allele is found in only a few (13Seldin M.F. D'Hoostelaere L.A. Steinberg A.D. Saga Y. Morse H.C.D. Immunogenetics. 1987; 26: 74-78Crossref PubMed Scopus (15) Google Scholar). The CD45.1 and CD45.2 alleles are distinguished antigenically by their reactivity to specific monoclonal antibodies, and the nucleotide changes that produce the antigenic differences have been identified (3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar). CD45 is encoded by a single gene, and expression appears to be regulated at the level of transcription (14Raschke W.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 161-165Crossref PubMed Scopus (19) Google Scholar). The murine gene is characterized by 34 exons and a large 50-kilobase (kb)1 intron between exons 2 and 3. CD45 transcription can initiate at three distinct sites in exon 1a, exon 1b, and downstream of exon 1b (15DiMartino J.F. Hayes P. Saga Y. Lee J.S. Int. Immunol. 1994; 6: 1279-1283Crossref PubMed Scopus (9) Google Scholar). However, the sequences responsible for the developmental and tissue-specific transcription of CD45 have yet to be identified. Extensive analyses have demonstrated that CD45 is required to generate signaling through both the T and B cell receptors (16Justement L.B. Campbell K.S. Chien N.C. Cambier J.C. Science. 1991; 252: 1839-1842Crossref PubMed Scopus (244) Google Scholar, 17Koretzky G.A. Picus J. Thomas M.L. Weiss A. Nature. 1990; 346: 66-68Crossref PubMed Scopus (391) Google Scholar, 18Koretzky G.A. Picus J. Schultz T. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2037-2041Crossref PubMed Scopus (326) Google Scholar, 19Lane P.J. Ledbetter J.A. McConnell F.M. Draves K. Deans J. Schieven G.L. Clark E.A. J. Immunol. 1991; 146: 715-722PubMed Google Scholar, 20Pingel J.T. Thomas M.L. Cell. 1989; 58: 1055-1065Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 21Weaver C.T. Pingel J.T. Nelson J.O. Thomas M.L. Mol. Cell. Biol. 1991; 11: 4415-4422Crossref PubMed Scopus (112) Google Scholar). In the case of antigen receptor signaling, CD45 is required for both the positive and negative regulation of the Src family kinases associated with the antigen receptors (22Thomas M.L. Brown E.J. Immunol. Today. 1999; 20: 406-411Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 23D'Oro U. Ashwell J.D. J. Immunol. 1999; 162: 1879-1883PubMed Google Scholar, 24Ashwell J.D. D'Oro U. Immunol. Today. 1999; 20: 412-416Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Katagiri T. Ogimoto M. Hasegawa K. Arimura Y. Mitomo K. Okada M. Clark M.R. Mizuno K. Yakura H. J. Immunol. 1999; 163: 1321-1326PubMed Google Scholar). Mice in which the expression of CD45 is impaired (26Kishihara K. Penninger J. Wallace V.A. Kundig T.M. Kawai K. Wakeham A. Timms E. Pfeffer K. Ohashi P.S. Thomas M.L. Furlonger C. Paige C.J. Mak T.W. Cell. 1993; 74: 143-156Abstract Full Text PDF PubMed Scopus (457) Google Scholar) or absent (27Byth K.F. Conroy L.A. Howlett S. Smith A.J. May J. Alexander D.R. Holmes N. J. Exp. Med. 1996; 183: 1707-1718Crossref PubMed Scopus (354) Google Scholar, 28Mee P.J. Turner M. Basson M.A. Costello P.S. Zamoyska R. Tybulewicz V.L. Eur. J. Immunol. 1999; 29: 2923-2933Crossref PubMed Google Scholar) have greatly reduced numbers of peripheral T cells, suggesting that CD45 is required for T cell development (26Kishihara K. Penninger J. Wallace V.A. Kundig T.M. Kawai K. Wakeham A. Timms E. Pfeffer K. Ohashi P.S. Thomas M.L. Furlonger C. Paige C.J. Mak T.W. Cell. 1993; 74: 143-156Abstract Full Text PDF PubMed Scopus (457) Google Scholar, 27Byth K.F. Conroy L.A. Howlett S. Smith A.J. May J. Alexander D.R. Holmes N. J. Exp. Med. 1996; 183: 1707-1718Crossref PubMed Scopus (354) Google Scholar). In contrast to T cell maturation, there appears to be little effect on B cell development in the null mice (26Kishihara K. Penninger J. Wallace V.A. Kundig T.M. Kawai K. Wakeham A. Timms E. Pfeffer K. Ohashi P.S. Thomas M.L. Furlonger C. Paige C.J. Mak T.W. Cell. 1993; 74: 143-156Abstract Full Text PDF PubMed Scopus (457) Google Scholar, 27Byth K.F. Conroy L.A. Howlett S. Smith A.J. May J. Alexander D.R. Holmes N. J. Exp. Med. 1996; 183: 1707-1718Crossref PubMed Scopus (354) Google Scholar). The role of CD45 in signaling in other lymphoid lineages is less well defined, although reports suggest that the CD45 phosphatase activity is important in regulating signal transduction in mast cells through the high affinity receptor for IgE, FcεRI (29Berger S.A. Mak T.W. Paige C.J. J. Exp. Med. 1994; 180: 471-476Crossref PubMed Scopus (71) Google Scholar, 30Murakami K. Sato S. Nagasawa S. Yamashita T. Int. Immunol. 2000; 12: 169-176Crossref PubMed Scopus (18) Google Scholar). Much less is known about the functional significance of the extracellular domain, although the isoform differences most likely convey ligand specificity. Studies have shown that the isoforms differentially affect activation through the T cell receptor (31Novak T.J. Farber D. Leitenberg D. Hong S.C. Johnson P. Bottomly K. Immunity. 1994; 1: 109-119Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 32Hall S.R. Heffernan B.M. Thompson N.T. Rowan W.C. Eur. J. Immunol. 1999; 29: 2098-2106Crossref PubMed Scopus (43) Google Scholar) and that the isoforms vary in their associations with the receptor (33Leitenberg D. Novak T.J. Farber D. Smith B.R. Bottomly K. J. Exp. Med. 1996; 183: 249-259Crossref PubMed Scopus (101) Google Scholar,34Leitenberg D. Boutin Y. Lu D.D. Bottomly K. Immunity. 1999; 10: 701-711Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The expression of specific isoforms has also been implicated in T cell apopotosis (35Ong C.J. Chui D. Teh H.S. Marth J.D. J. Immunol. 1994; 152: 3793-3805PubMed Google Scholar, 36Oka S. Mori N. Matsuyama S. Takamori Y. Kubo K. Immunology. 2000; 100: 417-423Crossref PubMed Scopus (14) Google Scholar, 37Renno T. Attinger A. Rimoldi D. Hahne M. Tschopp J. MacDonald H.R. Eur. J. Immunol. 1998; 28: 540-547Crossref PubMed Scopus (63) Google Scholar). Many of the CD45 expression experiments were conducted with cDNA transgenes flanked either by retroviral sequences (38Johnson P. Greenbaum L. Bottomly K. Trowbridge I.S. J. Exp. Med. 1989; 169: 1179-1184Crossref PubMed Scopus (59) Google Scholar) or by the thymocyte-specific proximal lck promoter and the human growth hormone minigene (39Chui D. Ong C.J. Johnson P. Teh H.S. Marth J.D. EMBO J. 1994; 13: 798-807Crossref PubMed Scopus (66) Google Scholar, 40Marth J.D. Ong C.J. Chui D. Adv. Exp. Med. Biol. 1994; 365: 149-166Crossref PubMed Scopus (3) Google Scholar). Expression from these CD45 cDNA constructs is not obtained consistently (41Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (651) Google Scholar), and in transgenic mice, the lck-controlled cDNAs only express 10–30% of endogenous CD45 protein levels (40Marth J.D. Ong C.J. Chui D. Adv. Exp. Med. Biol. 1994; 365: 149-166Crossref PubMed Scopus (3) Google Scholar). The 5′ and 3′ sequences used to regulate expression from these cDNA constructs differ considerably from those controlling the endogenous CD45 gene. The proximallck promoter is expressed in thymocytes but not in peripheral T cells (42Wildin R.S. Wang H.U. Forbush K.A. Perlmutter R.M. J. Immunol. 1995; 155: 1286-1295PubMed Google Scholar), a pattern of expression that is much restricted compared with endogenous CD45. In contrast, expression from the retroviral LTR promoter should be constitutive, but this construct lacks a discernible polyadenylation sequence in proximity to the CD45 cDNA. In addition, the 3′ sequences may contribute significantly to the expression of these cDNAs. Signals for controlling mRNA translation, stability, and localization have been found in the 3′-UTR of some genes, indicating that these sequences can contain key information for both positive and negative regulation of mRNA (43Decker C.J. Parker R. Curr. Opin. Cell Biol. 1995; 7: 386-392Crossref PubMed Scopus (220) Google Scholar,44Wickens M. Anderson P. Jackson R.J. Curr. Opin. Genet. Dev. 1997; 7: 220-232Crossref PubMed Scopus (281) Google Scholar). Therefore, the difficulty in consistently obtaining expression from these CD45 cDNA constructs may be due in part to the 5′ and 3′ regulatory elements used. To address the roles of these gene elements, we designed new CD45 cDNA vectors. Sections of the 3′-end of the CD45 gene downstream of the translational termination codon were evaluated in reporter gene constructs and in CD45 cDNA vectors. Regions of the 5′-end of the CD45 gene as large as 19 kb upstream of the initiation codon were tested for promoter activity and compared with the promoter for the human LFA-1 gene, a gene with similar expression characteristics to CD45. We also tested the effect of the introns between exons 3 and 9 in CD45 expression constructs. These introns flank the principal alternatively expressed exons 4–8. Here we report that by using these novel constructs, we are able to identify components that generate reproducible expression of CD45 in a variety of hematopoietic cell types. These results suggest that the CD45 intron sequences contain information necessary for expression from the transgenes. Furthermore, the intron sequences flanking the alternatively spliced exons provide correct leukocyte lineage-specific splicing. 70Z/3 is a methylnitrosourea-induced murine pre-B lymphoblast cell line. Transfection of 70Z/3 was performed by electroporation in cuvettes with a 3.5-mm gap. After adding 25 μg of linearized DNA to 1 × 107 cells in phosphate-buffered saline (PBS), the DNA-cell complex was kept on ice for 10 min and then placed in a BTX Transfector 300 (BTX, La Jolla, CA) for electroporation (settings: capacitance, 1400 microfarads; voltage, 200 V). Following electroporation, the samples were incubated on ice for 10 min and then resuspended in growth medium for 48 h. Cells were subsequently grown in medium containing 1.5 mg/ml G418 sulfate (G418; Omega Scientific, Tarzana, CA). MD.45–27J (27J) is a cytotoxic murine T cell hybridoma clone obtained from Joseph Lustgarten (Sidney Kimmel Cancer Center, La Jolla, CA) (45Gorochov G. Lustgarten J. Waks T. Gross G. Eshhar Z. Int. J. Cancer Suppl. 1992; 7: 53-57PubMed Google Scholar). Twenty micrograms of linearized plasmid DNA were mixed with 1 × 107 cells in PBS, and immediately electroporation (800 microfarads and 250 V) was performed. After electroporation, the cells were placed on ice for 10 min and then resuspended in growth medium for 36 h. Cells expressing the neomycin gene were selected using 2.0 mg/ml G418. BW5147, a murine thymic lymphoma cell line, was transfected using 1 × 107 cells in PBS and 25 μg of linearized plasmid DNA. The mixture of cells and DNA was incubated on ice for 10 min prior to electroporation (250 V and 2200 microfarads). After electroporation, the cells were placed on ice for 10 min and then resuspended in growth medium. After 48 h, the cells were placed in medium containing 1.0 mg/ml G418. Human embryonal kidney cells (293) were transfected using LipofectAMINE Reagent (Life Technologies, Inc.). One day before transfection, 1.2 × 106 cells were plated on 10-cm dishes treated with 0.1 mg/ml poly-d-lysine (Sigma). For transfection, 6 μg of supercoiled plasmid DNA, 40 μl of LipofectAMINE, and 300 μl of unsupplemented growth medium were mixed and kept for 15 min. at room temperature. After the 293 cells were washed two times with unsupplemented medium, the DNA/lipid mixture was added and incubated with the cells for 5 h at 37 °C. The cells were washed twice with supplemented medium and grown in 10 ml of medium for 48 h. S49 is a murine T lymphoma cell line provided by Robert Hyman (The Salk Institute, La Jolla, CA). Transfection was performed by electroporation (250 V, 1400 microfarads). The human T cell leukemia line, Jurkat, was obtained from Javi Piedrafita (Sidney Kimmel Cancer Center, La Jolla, CA). Jurkat was transfected using DMRIE-C reagent (Life Technologies) according to the manufacturer's directions. All cell lines were obtained from ATCC (Manassas, VA) unless otherwise indicated. The growth medium for the cell lines was Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 units/ml penicillin, and 10 μg/ml streptomycin except for 70Z/3, which was grown in RPMI 1640 medium supplemented with 2 mml-glutamine, 10 mm HEPES, 1.0 mmsodium pyruvate, 10% fetal bovine serum, 0.05 mm2-mercaptoetanol, 10 units/ml penicillin, and 10 μg/ml streptomycin. All media components were from Irvine Scientific (Irvine, CA). To study CD45 cDNA expression in a variety of leukocyte populations, we obtained three constructs. The plasmid, pARV/CD45 (H), was kindly provided by K. Bottomly (Yale University School of Medicine, New Haven, CT). This plasmid is a modification of pARV/CD45 (38Johnson P. Greenbaum L. Bottomly K. Trowbridge I.S. J. Exp. Med. 1989; 169: 1179-1184Crossref PubMed Scopus (59) Google Scholar) in which the neomycin gene was replaced with the hygromycin gene (31Novak T.J. Farber D. Leitenberg D. Hong S.C. Johnson P. Bottomly K. Immunity. 1994; 1: 109-119Abstract Full Text PDF PubMed Scopus (110) Google Scholar). Since selection for hygromycin resistance in leukocytes is inefficient, we removed the hygromycin gene as a ClaI fragment and reinserted the neomycin gene (GKneo), creating pARV/CD45 (N). The neomycin gene from pGKneocbpA (Eva Lee, University of California, San Diego, La Jolla, CA) was used and contains the mammalian phosphoglycerate kinase promoter and the bovine growth hormone poly(A) sequence. The CD45 cDNA plasmids, pML84 and pML171, were obtained from J. Marth (Department of Medicine and Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA). The full-length cDNA is present in pML84 (CD45RABC), while pML171 carries the CD45 cDNA encoding the isoform missing exons 4–6 (CD45RO). In these vectors, the transgenes are inserted 3′ of the proximal lck promoter and upstream of the human growth hormone gene (40Marth J.D. Ong C.J. Chui D. Adv. Exp. Med. Biol. 1994; 365: 149-166Crossref PubMed Scopus (3) Google Scholar). We added the GKneo NotI fragment into the NotI site of each plasmid to facilitate selection of stable transfectants. In all of the cDNA transgenes, the 3200-base pair (bp) XbaI fragment containing the CD45.2 allelic sequence was replaced with the corresponding XbaI fragment specifying the sequences for the CD45.1 allele. To test the 3′ CD45 sequences, the plasmids pSPORT-(−A), pSPORT-BGA, pSPORT-XR, and pSPORT-EA, were generated (Fig. 1 A). These plasmids are derivatives of pCMVSPORTβgal (Life Technologies). The SV40 small t/poly(A) site of pCMVSPORTβgal was removed creating pSPORT-(−A) (Fig. 1 A). The SV40 small t/poly(A) was replaced by a 280-bp XbaI to XhoI bovine growth hormone poly(A) insert from pGKneocbpA to produce pSPORT-BGA (Fig. 1 A). An 868-bp XbaI toEcoRI piece from CD45 exon 33 was introduced into pSPORT-(−A), generating pSPORT-X (not shown). The XbaI site lies 15 bp downstream from the CD45 stop signal, and theEcoRI site is 165 bp upstream from the 3′-end of the CD45 mRNA (3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar). An adjacent 800-bp EcoRI fragment containing the remaining 3′ portion of exon 33 and all CD45 polyadenylation signals were cloned into pSPORT-X, resulting in pSPORT-XR (Fig.1 A). This EcoRI fragment also was introduced into pCMVSPORTβgal upstream from the SV40 small t/poly(A) creating pSPORT-EA (Fig. 1 A). To study the effect of 5′ sequences on CD45 cDNA expression, the constructs pLFAT200 and pCMVT200 (Fig. 1 B) were made by subcloning all components into pBluescript KS (Stratagene, La Jolla, CA). The 5′ cDNA from T200/3/pAX (38Johnson P. Greenbaum L. Bottomly K. Trowbridge I.S. J. Exp. Med. 1989; 169: 1179-1184Crossref PubMed Scopus (59) Google Scholar) containing all alternatively spliced exons (exons 4–8) was inserted as aClaI/XbaI fragment. The 3′ CD45 DNA was subcloned from pSPORT-XR as a 1800-bp XbaI/EcoRI fragment (striped box in Fig. 1 A). The remaining cDNA region obtained from a CD45.1 allele (3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar) extended from exon 9 through 33 and was inserted as a 3200-bp XbaI fragment. A 1800-bp SalI/ClaI fragment containing the human leukocyte function-associated antigen (LFA-1, CD11a) promoter was inserted 5′ of the CD45 cDNA sequences. The LFA-1 promoter was obtained by PCR amplification from DNA isolated from the human T lymphocyte line, Jurkat. The sense primer extended from position −1694 to −1668 of the LFA-1 promoter (46Cornwell R.D. Gollahon K.A. Hickstein D.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4221-4225Crossref PubMed Scopus (41) Google Scholar) and contained uniqueSalI and SfiI sites at the 5′-end. The antisense primer extended from +77 to +96 of the LFA-1 promoter (46Cornwell R.D. Gollahon K.A. Hickstein D.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4221-4225Crossref PubMed Scopus (41) Google Scholar) and had unique ClaI and SgfI sites engineered at the 3′-end of the LFA-1 sequence. The construct, pLFAT200 (Fig.1 B), was completed by the addition of the neomycin gene (GKneo). The LFA promoter was replaced with the cytomegalovirus (CMV) promoter to generate pCMVT200 (Fig. 1 B) by digestion of pLFAT200 with SfiI and ClaI and insertion of aNruI/HindIII fragment from pcDNA3 (Invitrogen, Carlsbad, CA). To investigate regulatory sequences within the introns, the CD45 minigene construct, pLFAiT200 (Fig. 1 C), was made by inserting all components into a modified mammalian expression vector pcDNA3 in which the CMV promoter had been replaced by the 1800-bp fragment containing the LFA-1 promoter. The 5′ CD45 cDNA/intron region in LFAiT200 starts in exon 1b at an NheI site, 27 bp from the beginning of the exon, continues through exons 2 and 3, and ends within the 3/4 intron at a BglII site, 500 bp downstream from exon 3. Two PCR products were used to generate this region. PCR amplification of cDNA extending from exon 1b to 3 produced the first fragment, and amplification of CD45 genomic DNA containing exon 3 and intron sequences 3′ to exon 3 resulted in the second. The fragment was completed by ligating the two PCR products. A λ genomic clone carrying CD45 exons 3–8 was used to obtain aBglII to BamHI fragment containing exon 4. This fragment was inserted downstream from exon 3 at the BglII (intron) and BamHI (vector) sites. The genomic sequences, extending from the BamHI site 550 bp upstream of exon 5 to an XhoI site 600 bp downstream of exon 8, were taken from a λ genomic clone, placed into the plasmid vector pGEM11Z (Promega, Madison, WI) and subsequently transferred to the minigene construct as a BamHI/NotI fragment generating plasmid pLFA1–8. The genomic region containing exons 8 and 9 was amplified by PCR from mouse genomic DNA and subcloned as a 2000-bpHindIII/EcoRI fragment into pGEM11Z, creating pG11(8–9). For this amplification, the 35-bp sense oligonucleotide primer was the complete exon 8 sequence, and the antisense primer extended from base 192 to 215 of exon 9. The PCR amplification fragment included the XhoI site downstream from exon 8 and theXbaI restriction site within exon 9. The XbaI toEcoRI 3′ CD45 DNA fragment in pSPORT-XR was inserted into pG11(8–9) as an XbaI/NotI fragment generating pG11(8–9)A. A CD45.1 cDNA region (3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar) extending from exon 9 to 33 was subcloned into pG11(8–9)A as an XbaI fragment producing pG11(8-A). The CD45 minigene construct, pLFAiT200, was completed by the introduction of the XhoI/NotI restriction fragment from pG11(8-A) into pLFA1–8. The final construct contains CD45 sequence consisting of a cDNA 5′ region (exons 1b–3), a genomic DNA region extending from exon 3 to exon 9, a cDNA segment from exon 9 to exon 33, and 3′ gene sequences from exon 33 to 750 bp downstream of the end of transcriptional termination. The potential promoter region upstream of exon 1a was isolated from a λ genomic library constructed in the LambdaGEM-12 cloning vector (Promega). The longest λ clone extended ∼19 kb upstream and 1800 bp downstream of the CD45 translational start site. The insert was flanked by NotI sites from the vector. A partial restriction map of a subsection of this DNA is shown in Fig. 1 D; theNheI site maps within exon 1b. The entire upstream region was subcloned into plasmid pGEM5Z as three smaller fragments: a 15-kbNotI/PstI fragment consisting of the most upstream 5′ sequences (pG5NP); a 1.3-kb PstI fragment (pG5P); and a 4.5 kb, PstI/NotI fragment including exons 1a, 1b, and 2 as well as 1.7 kb of intron sequence 3′ of exon 2 (pG5PN). The 5′ NotI site was changed to aSfiI site using double-stranded oligonucleotides containing a NotI overhang and an internal SfiI site. Using the natural EcoRI, PstI, and NheI sites and the synthetic SfiI site, the following plasmids were generated: pCD45FiT200, pCD45EiT200, pCD45PiT200, and pCD45P2iT200 (Fig. 1 D). Total RNA was purified from cells using TRIZOL (Life Technologies) following the manufacturer's directions. Fifty micrograms RNA isolated from cells transfected with plasmids pLFAT200 and pCMVT200 were treated with 1 unit of DNase I (Promega) for 1 h at 37 °C. The DNase I was removed by one extraction with phenol/chloroform/isoamyl alcohol followed by one extraction with chloroform/isoamyl alcohol as suggested by Promega. The RNA was precipitated with ethanol and resuspended in RNase-free water. RT-PCR was performed using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) to distinguish transgene CD45.1 mRNA transcripts from the endogenous CD45.2 transcripts of the transfected cells. All RT and PCR reactions were assembled according to the manufacturer's recommendations. The antisense oligonucleotide used for cDNA synthesis, 5′-GAGACCAGAAACTCATAG-3′, contains the last 13 bp of exon 14 and the first 5 bp of exon 15. For the PCR component, the sense oligonucleotide primer was the complete exon 3 sequence, 5′-GGCAAACACCTACACCCAGTGATG-3′. The antisense oligonucleotides are specific for the CD45.1 (Ly5 a) and CD45.2 (Ly5 b) alleles (3Zebedee S.L. Barritt D.S. Raschke W.C. Dev. Immunol. 1991; 1: 243-254Crossref PubMed Scopus (17) Google Scholar, 47Raschke W.C. Hendricks M. Chen C.M. Immunogenetics. 1995; 41: 144-147Crossref PubMed Scopus (12) Google Scholar) and consist of bases 86–106 of exon 12. The sequences of these antisense primers are as follows with the allele-specific bases underlined: CD45.1, 5′-CCATGGGGTTTAGATGCAGGA-3′ and CD45.2, 5′-CCATGGGGTTTAGATGCAGAC-3′. The cDNA was synthesized for 1 h at 42 °C followed by 5 min at 95 °C. The cDNA was amplified for 34 cycles. The first cycle consisted of 2 min at 94 °C, 1 min at 63 °C, and 3 min at 72 °C. The remaining cycles were 1 min at 94 °C, 1 min at 63 °C, and 3 min at 72 °C. The amplified products were separated on a standard TBE-agarose gel (48Sambrook J. Fritsch E.F. Maniatis T. Molecular Clonin" @default.
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• W2092003249 date "2001-01-01" @default.
• W2092003249 modified "2023-10-15" @default.
• W2092003249 title "The Role of Intron Sequences in High Level Expression from CD45 cDNA Constructs" @default.
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• W2092003249 doi "https://doi.org/10.1074/jbc.m100448200" @default.
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