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WO2006070729A1 - Promoteur specifique d'une cellule produisant de l'insuline et son utilisation - Google Patents

Promoteur specifique d'une cellule produisant de l'insuline et son utilisation Download PDF

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Publication number
WO2006070729A1
WO2006070729A1 PCT/JP2005/023758 JP2005023758W WO2006070729A1 WO 2006070729 A1 WO2006070729 A1 WO 2006070729A1 JP 2005023758 W JP2005023758 W JP 2005023758W WO 2006070729 A1 WO2006070729 A1 WO 2006070729A1
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Prior art keywords
promoter
insulin
seq
cells
vector
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Ceased
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PCT/JP2005/023758
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English (en)
Japanese (ja)
Inventor
Noriaki Tanaka
Naoya Kobayashi
Takuya Fukazawa
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Okayama University NUC
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Okayama University NUC
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Priority to JP2006550751A priority Critical patent/JP4825978B2/ja
Publication of WO2006070729A1 publication Critical patent/WO2006070729A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the present invention relates to a novel insulin-producing cell-specific promoter and use thereof.
  • a plasmid containing a promoter region involved in expression of the target protein and a reporter gene downstream from the cell population containing cells expressing the target protein is used in the Balta cell population.
  • This is a method in which a reporter gene expression positive cell is selected using a cell sorter or an antibiotic resistance gene (for example, see Soria et al., Diabetologia (2001) 44: 407-415).
  • This method is applicable to the selection of insulin-producing cells.
  • the natural promoter sequence is used as it is, the specificity decreases due to the non-specific transcription active site of insulin-producing cells, and the selection of cells is more strict. I can't do it.
  • An object of the present invention is to provide a novel promoter with high specificity and strong transcriptional activity in order to efficiently select insulin-producing cells.
  • the 363rd 5 'flanking region (from SEQ ID NO: 1, hereinafter,-363/1 1) upstream of the transcription start site (+1) of the human insulin promoter (+1) ⁇ 363 / —1 area, including the Karakcho Hacchohachi box, and 3 areas deleted _363 / _32 areas (SEQ ID NO: 2) are inserted into the ⁇ side of the The present inventors have found that a novel promoter with high specificity and high transcriptional activity can be obtained in production cells.
  • the present invention provides: a) a polynucleotide containing the base sequence represented by SEQ ID NO: 2 on the side of the base sequence represented by SEQ ID NO: 1, or
  • the present invention also relates to a vector comprising the promoter.
  • a reporter gene and / or an antibiotic resistance gene is preferably linked to the upstream side of the promoter.
  • the present invention further includes (a) introducing a vector having a reporter gene and an antibiotic resistance gene linked to the 3 'side of the promoter into undifferentiated cells,
  • the present invention relates to a method for selecting insulin-producing cells comprising
  • FIG. 1 is a diagram showing the binding sites of major transcription factors of the natural human insulin promoter.
  • FIG. 2 is a diagram showing an embodiment of the method for selecting insulin-producing cells of the present invention.
  • FIG. 3 is a diagram showing an embodiment of the method for selecting insulin-producing cells of the present invention.
  • FIG. 4 shows an embodiment of subcloning of the present invention.
  • FIG. 5 Electrophoresis photograph. M indicates molecular weight marker, lanes:!
  • To 6 restriction enzymes of pGL3, phlNS—363 / —1 ⁇ Luc, phlNS—363 / —1 + (hINS—363 / —32) XI to 4-Luc, respectively Processed by Hindlll and Kpnl.
  • FIG. 6 is a graph showing the promoter activity of each plasmid in each cell under low-gnorecose conditions.
  • FIG. 7 is a graph showing the promoter activity of each plasmid in each cell under high-gnorecose conditions.
  • the promoter of the present invention includes: a) a polynucleotide containing the base sequence represented by SEQ ID NO: 2 on the ⁇ side of the base sequence represented by SEQ ID NO: 1, or b) the base sequence of the polynucleotide of a) above And a polynucleotide having a promoter activity in insulin-producing cells, which hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions.
  • the nucleotide sequence of SEQ ID NO: 1 represents the _ 1 to _ 363th flanking region (one 363 / -1 region) upstream of the transcription start site (+1) of the natural human insulin promoter. It has a binding site for transcription factors such as PDX1, NeuroD, NKX2.2, NKX6.1 and HNF4a and a C1-A2 region responsible for glucose-dependent transcriptional activity (Figure 1) (German et al., J. Biol. Chem). , 1996, Bartoov—Shifman et al., J. Biol. Chem., 2002, Watada et al., Proc. Natl. Acad.
  • the base sequence of SEQ ID NO: 2 is a region (_363 / _32) excluding the base sequence ability TATA box of SEQ ID NO: 1.
  • the promoter of the present invention further has the base sequence of SEQ ID NO: 2 inserted in tandem.
  • the number of the nucleotide sequence represented by SEQ ID NO: 2 contained in the promoter of the present invention is not particularly limited, but:! To 15 is preferable:! To 4 is more preferable 2 is the most preferable. Only the nucleotide sequence represented by SEQ ID NO: 1 cannot select an efficient insulin cell with low promoter activity. Also represented by SEQ ID NO: 2. If the number of base sequences to be transferred is 16 or more, the total molecular weight becomes too large when inserted into a vector, and the efficiency of introduction into cells tends to decrease.
  • in tandem means repeatedly inserting the same sequence in the same direction.
  • the promoter of the present invention can generally be produced by a PCR subcloning method or the like. For example, using the human genome as a saddle, the nucleotide sequence of SEQ ID NO: 1 is amplified by PCR and inserted into an appropriate vector. Next, the base sequence of SEQ ID NO: 2 is amplified using this vector as a cage. There is a method for producing a desired promoter by inserting an arbitrary number of the nucleotide sequence of SEQ ID NO: 2 upstream (5 ′ side) of the vector into which the nucleotide sequence of SEQ ID NO: 1 is inserted.
  • an additional base sequence such as a restriction enzyme cleavage site is inserted between these base sequences. May be.
  • an additional base sequence such as a restriction enzyme cleavage site is inserted between these base sequences. May be.
  • nucleotide sequences of SEQ ID NOS: 1 and 2 several bases are deleted / replaced / inserted except in the TATA box or the binding site of the main transcription factor unless the promoter activity of the present invention is reduced. are also included in the promoter of the present invention.
  • several bases may be deleted, substituted or inserted as long as they do not interfere with the binding of TATA box binding factors or transcription factors. It is included in the promoter of the invention.
  • stringent conditions means conditions for hybrid formation and subsequent washing.
  • a high ion concentration for example, 6 X SSC
  • a solution containing 1.5M NaCl and 0.15M trisodium citrate is set to 10 X SSC
  • a hybrid is formed at 45 ° C in a solution containing 50% formamide under a low ion concentration (for example, , 2 X SSC) at 50 ° C or under high ion concentration (eg, 6 X SSC) solution at 65 ° C to form a hybrid and under low ion concentration (eg 0 1 X SSC) can be used for cleaning at 65 ° C.
  • the salt concentration in the cleaning step should be selected from 2 X SSC (low stringency conditions) to 0.2 X SSC (high stringency conditions) force, for example, when the temperature is set to 50 ° C. Can do.
  • the temperature in the washing step is, for example, room temperature (low stringency conditions)
  • Hybridization is "Molecular Cloning 3rd Edition", “Current 'Protoco One Norez' In 'Molecular' Biology”, “DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford According to the method described in “University (1995)”, colony 'hybridization method, plaque' hybridization method or Southern blot hybridization method can be performed. .
  • Polynucleotides that can hybridize under stringent conditions include NCBI (National Center for Biotechnology).
  • any plasmid vector is preferred. Any vector can be used as long as any promoter of the original vector is excised by restriction enzyme treatment. Further, those having a plurality of restriction enzyme sites for inserting foreign genes are preferred. Examples of such vectors include a reporter gene, which has no promoter upstream and has a plurality of restriction enzyme recognition sites, and is originally prepared for reporter assembly of any promoter.
  • PGL2 basic vector Promega
  • pGL3 basic vector Promega
  • pGL4 basic vector Promega
  • a reporter gene is linked to the downstream side of the promoter of the present invention. Considering the transfection into cells that are preferred, it is preferable that the final vector size is about 1 OKbp or less.
  • the most commonly used method is the luciferase atsey method.
  • the luciferase atsey method can measure even very fine activities and is a simple method, so it is preferable for the purpose of activity evaluation.
  • Such a reporter gene is not particularly limited as long as the detection method of the gene product is known, for example, an enzyme gene such as a luciferase gene and a chloramphenicol acetyltransferase (CAT) gene, green Fluorescent protein genes such as fluorescent protein (GFP) gene and red fluorescent protein (DS-RED) gene can be used.
  • an enzyme gene such as a luciferase gene and a chloramphenicol acetyltransferase (CAT) gene
  • green Fluorescent protein genes such as fluorescent protein (GFP) gene and red fluorescent protein (DS-RED) gene
  • GFP fluorescent protein
  • DS-RED red fluorescent protein
  • an antibiotic resistance gene since the promoter of the present invention functions specifically in insulin-producing cells, an antibiotic resistance gene must be linked downstream of the 3 'side to select insulin-producing cells. Furthermore, it is preferable to use a reporter gene and an antibiotic resistance gene linked together.
  • Antibiotic resistance genes used in the present invention are useful, such as nodal, idalomycin resistance gene, neomycin resistance gene, zeocin resistance gene, bradysidine resistance gene, etc., and may be more appropriate depending on the cell type and selection design. One can be selected as appropriate. In addition, other resistance genes from other promoters can be linked to the vector of the present invention for different selection.
  • GFP and hygromycin resistance gene fusion gene GFP and hygromycin resistance gene fusion gene (GFP_HygroR fusion) or RED and hygromycin resistance gene fusion.
  • a reporter gene such as a gene (DS-RED-HygroR fusion) and an antibiotic resistance gene fusion can also be linked.
  • the reporter gene can be easily detected with a fluorescence microscope.
  • the gene encoding green fluorescent protein or red fluorescent protein is preferred because it can be detected and color is easily recognized.
  • a method for linking such a reporter gene, an antibiotic resistance gene, or a fusion gene of a reporter gene and an antibiotic resistance gene (hereinafter also referred to as a fusion gene) to the promoter of the present invention is known in the art.
  • Various methods can be used.
  • the reporter assembly vector as described above is used to produce the promoter of the present invention, the reporter gene present in the vector is replaced with the reporter gene, antibiotic resistance gene or fusion gene to be ligated. Can do.
  • the luciferase gene can be excised with two restriction enzymes, Ncol and Xbal, and the target gene can be obtained by using a primer that adds Ncol and Xbal cleavage sites, respectively.
  • the gene can be exchanged by amplifying the RFRF by PCR and ligating the insert fragment treated with the restriction enzyme.
  • a ribofusion method As a method for introducing the obtained vector into a cell, a ribofusion method, an electopore position method, a calcium phosphate method, or the like can be used. If the size of the vector produced exceeds lOkbp, or if the target cells are cells that are difficult to enter by the lipofusation method, such as ES cells, mesenchymal stem cells, and bone marrow cells, the electoral position method is preferred.
  • the promoter of the present invention specifically functions in insulin-producing cells, it can be used for selection of insulin-producing cells.
  • the promoter of the present invention containing n base sequences represented by SEQ ID NO: 2 on the 5 ′ side of the base sequence represented by SEQ ID NO: 1: phINS—363 / —l + (hINS—363 / — 32) Plasmid vector ligated with a gene encoding green fluorescent protein (GFP) fused with hygromycin resistance gene upstream of X n: pMNS _ 363 / _ l + (MNS— 363 /-32) X n 'GFP—Build HygroR.
  • GFP green fluorescent protein
  • Undifferentiated cells such as human ES cells or mesenchymal stem cells are cultured and proliferated, and an optimal amount of the plasmid is introduced by electoporation. Differentiation is induced after exchanging the culture medium. Insulin-producing cells into which the plasmid has been introduced develop a green color under a fluorescence microscope, so when observed over time, hygromycin is added to the culture solution when the percentage of green cells and the fluorescence intensity become maximum. Insulin-producing cells can be obtained by selecting GFP-positive cells ( Figure 2).
  • plasmid pMNS— 363 / — 1 + (M NS-363 /-32) X n 'GFP— HygroR / SV40' Neo can be used.
  • plasmid cells into which the plasmid has been introduced can be selected by neomycin treatment before induction of differentiation into insulin-producing cells (Fig. 3).
  • the antibiotic resistance gene to be used can be changed as appropriate to the above-mentioned antibiotic resistance genes other than hygromycin and neomycin, and the reporter gene can also be changed appropriately to the above-mentioned genes other than GFP. it can.
  • the optimal amount for introducing the plasmid is preferably 2 ⁇ g to 20 / g of the plasmid for 2 ⁇ 10 7 to 5 ⁇ 10 7 cells.
  • Differentiation induction is preferably carried out at a high gno-lecose concentration, and the culture period is preferably 2 to 6 weeks.
  • Human genome l OOng purified from a human normal lung fibroblast cell line (NHLF) from the _ 1 to _ 363 th 5 r flanking region (SEQ ID NO: 1) upstream of the transcription start site (+1) of the human insulin promoter
  • the reaction conditions (94.C, 15 ° C, 62.5 ° C, 30 ° C, 68 ° C, 1 min.) PCR product 1 (hereinafter referred to as Xhol / 363 / -l) / HindIII).
  • forward primer 1 (SEQ ID NO: 3) incorporating an Xhol cleavage site on the 5 ′ side and reverse primer 1 (SEQ ID NO: 4) incorporating a Hindlll cleavage site on the 3 ′ side were used for the human insulin promoter 363 / — Set at both ends of one area.
  • PCR Table 1 shows the composition of the reaction solution.
  • the total amount of PCR product 1 (Xhol / —363 / —1 / Hindlll) obtained in (A) above was treated with restriction enzymes Xhol and Hindlll at 10 U for 16 hours at 37 ° C.
  • the luciferase expression plasmid pGL3 basic vector (Promega) 5 was treated with the same two restriction enzymes 15 U for 3 hours at 37 ° C for 3 hours, and then the Quick ligation kit (M220 OS: Ligation was performed using New England Biolab (Fig. 4).
  • ligation For ligation, mix 3 times the amount of insert DNA in a molar ratio with 50 ng of vector DNA, dilute with nucleic acid-free water to make 10 ⁇ ⁇ , and use the 2 X Quick Ligation Buffer supplied with the kit and 1 ⁇ l. This was performed by adding quill quick T4DNA ligase and incubating at room temperature for 5 minutes. Of the obtained ligation product, ⁇ ⁇ was added to a 100 a 1 competent cell (DH5a, Toyobo Co., Ltd.) placed on ice for 30 minutes after dissolution.
  • DH5a a 1 competent cell
  • the obtained plasmid was treated with Xhol and Hindlll, and it was confirmed after electrophoresis on a 1% agarose gel containing ethidium bromide that the target sequence was cut out as an insert (Fig. 5, lane 2). 1 (hereinafter also referred to as phINS_ 363 / _ l 'Luc)
  • Has a cleavage site—363 / —32 regions as PCR products 2 to 5 (hereinafter referred to as Smal / —363 / —32 / XhoI, Nhel / —363 / —32 / SmaI, Mlul / -363 / — 32 / NheI and KpnlZ—363Z—32ZMluI.
  • Plasmid 1 phlNS—363 / —1'Luc obtained in (B) and PCR product 2: Smal / —363 / —32 / XhoI obtained in (C) were used with restriction enzymes Smal and Xhol. After the treatment, ligation was performed in the same manner as in (B) below, and the target of incorporating one 363 / -32 region into the Smal and Xhol sites of phlNS—363 / —l 'Luc. Plasmid 2 (also referred to as phINS (363 / -32) X l 'Luc) was obtained.
  • reporter assembly was performed as follows.
  • pCMV j3 -gal (6 ⁇ g) 1.
  • Opti_men ribofunction-specific culture medium Opti_men (Gibco BRL)
  • a negative control a mixture of pCMV j3—gal ⁇ z g) and pGL3 basic vector (6 ⁇ g) was used.
  • ribofusion reagent (manufactured by Falcon) was added, and Opti-men was added to the total volume of 300 ⁇ 1. It was. After standing for 45 minutes, each plasmid in the small tube was added to the clear tube together with the medium, mixed with the ribofecation reagent, and allowed to stand for another 15 minutes.
  • a serum-containing culture solution (Dulbecco's modified eagle medium (DMEM) supplemented with 2 mM or 16 mM L-glucose) (2.4 ml) was added to a total volume of 3.0 ml, and mixed well to prepare a lipofussion preparation.
  • DMEM Dulbecco's modified eagle medium
  • the medium was renewed (2 ml). There are a total of 7 combinations of pCMV iS -gal + various plasmids to perform this experiment, each cell requires a total of 24 tools (4 on a 6-wall plate), and experiments with low glucose concentrations This multiple was used to divide (2 mM) and high glucose concentration (16 mM), ie a total of 48 tools (eight on a 6 tool plate).
  • Table 2 shows the test cells and their origins.
  • Test plasmids are shown in Table 3.
  • RLUZ J3 gal value of each well was measured, and the average value of RLU / / 3—gal value of 3 tools of pGL3 + pCMV / 3-gal used as negative control was calculated, and this value was multiplied by fold value. 1.
  • the RLUZ j3_gal value of each tool when 0 was set was calculated. Fold Induction in each combination: Luc (RLU) / / 3—Calculate the mean and SD from the gal value. Analysis was performed.
  • Plasmids 2 to 6 showed no activity in human cells derived from other germ layers other than the inner lung lobe (MCF7 and 293T). Similarly, no activity was observed in other endoderm-derived cells except ⁇ -cells, including endoderm-derived sputum cancer cells (MIAPACA2) and a-cell-derived gonoregonal gon-producing cells (e.g., TC1). ⁇ 6, a ⁇ -cell-derived insulin-producing cell, exhibited 4.3-fold promoter activity in plasmid 1 and gradually increased in plasmids 2-5 (74-4-102.8-fold) under low glucose conditions. (Figure 6). Furthermore, these activities were further enhanced under high glucose conditions (FIG. 7). Industrial applicability
  • the promoter of the present invention shows very strong specificity to insulin-producing cells and has high transcriptional activity, for example, downstream of the promoter of the present invention, reporter genes such as luciferase and GFP, and antibiotic resistance genes
  • reporter genes such as luciferase and GFP, and antibiotic resistance genes
  • the promoter of the present invention is responsive to glucose concentration, and can be selected more strictly for cell populations having insulin promoter activity by culturing under high-concentration glucose conditions.
  • SEQ ID NO: 3 Forward primer incorporating a Xhol restriction site on the ⁇ side of the region of the human insulin promoter — 363 / — 1
  • SEQ ID NO: 4 Hindlll restriction site on the: ⁇ side of the human insulin promoter — 363 / —1 region Reverse primer incorporating position
  • SEQ ID NO: 5 Forward primer incorporating a Smal restriction site on the ⁇ side of the 363 / -32 region of the human insulin promoter
  • SEQ ID NO: 6 Reverse primer incorporating the Xhol restriction site on the: ⁇ side of the —363 / —32 region of the human insulin promoter
  • SEQ ID NO: 7 Forward primer incorporating a Nhel restriction site 5 ′ of the 363 / _32 region of the human insulin promoter
  • SEQ ID NO: 8 Reverse primer incorporating a Smal restriction site on the: ⁇ side of the -363 / -32 region of the human insulin promoter
  • SEQ ID NO: 9 Forward primer incorporating a Mlul restriction site 5 ′ of the human insulin promoter — 363 / _32 region
  • SEQ ID NO: 10 Reverse primer incorporating a Nhel restriction site on the ⁇ side of the -363 / -32 region of the human insulin promoter
  • SEQ ID NO: 11 Forward primer incorporating a Kpnl restriction site 5 'to the 363 / -32 region of the human insulin promoter
  • SEQ ID NO: 12 Reverse primer incorporating a Mlul restriction site 3 'to the 363 / -32 region of the human insulin promoter

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Abstract

Afin de sélectionner efficacement une cellule β humaine qui produit de l'insuline, l'invention se propose de mettre au point un nouveau promoteur ayant une spécificité élevée et une forte activité transcriptionnelle. Le promoteur comprend a) un polynucléotide dans lequel une séquence de bases représentée par SEQ ID NO: 2 est située du côté 5’ d'une séquence de bases représentée par SEQ ID NO: 1, ou un polynucléotide qui est hybridé à un polynucléotide composé d'une séquence de bases complémentaire de la séquence de bases du polynucléotide selon a) dans des conditions stringentes et présente une activité promotrice dans une cellule produisant de l'insuline.
PCT/JP2005/023758 2004-12-27 2005-12-26 Promoteur specifique d'une cellule produisant de l'insuline et son utilisation Ceased WO2006070729A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017015497A3 (fr) * 2015-07-21 2017-06-08 Indiana University Research & Technology Corporation Adn méthylé et déméthylé acellulaire dans les maladies résultant d'anomalies du taux de glucose dans le sang

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BARTOOV-SHIFMAN R. ET AL.: "Activation of the Insulin Gene Promoter through a Direct Effect of Hepatocyte Nuclear Factor 4", J. BIOL. CHEM., vol. 277, no. 29, July 2002 (2002-07-01), pages 25914 - 25919, XP002999680 *
FANG R.-X. ET AL.: "Multiple cis Regulatory Elements for Maximal Expression of the Cauliflower Mosaic Virus 35S Promoter in Transgenic Plants", PLANT CELL, vol. 1, no. 1, January 1989 (1989-01-01), pages 141 - 150, XP002999683 *
GERMAN M. ET AL.: "The Insulin Gene Promoter: A Simplified Nomenclature", DIABETES, vol. 44, 1995, pages 1002 - 1004, XP008067042 *
HARRINGTON R. H. ET AL.: "Transcription Factors Recognizing Overlapping C1-A2 Binding Sites Positively Regulate Insulin Gene Expression", J. BIOL. CHEM., vol. 276, no. 1, January 2001 (2001-01-01), pages 104 - 113, XP002999682 *
LAY J.L. ET AL.: "Identification of a Novel PDX-1 Binding Site in the Human Insulin Gene Enhancer", J. BIOL. CHEM., vol. 279, no. 21, 21 May 2004 (2004-05-21), pages 22228 - 22235, XP002999681 *
ODAGIRI H. ET AL.: "Function of the Human Insulin Promoter in Primary Cultured Islet Cells", J. BIOL. CHEM., vol. 271, no. 4, January 1996 (1996-01-01), pages 1909 - 1915, XP002999679 *
SORIA B. ET AL.: "From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus", DIABETOLOGIA, vol. 44, no. 4, April 2001 (2001-04-01), pages 407 - 415, XP002183386 *
SORIA B. ET AL.: "Insulin-Secreting Cells Derived From Embryonic Stem Cells Normalize Glycemia in Streptozotocin-Induced Diabetic Mice", DIABETES, vol. 49, no. 2, February 2000 (2000-02-01), pages 157 - 162, XP002183378 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017015497A3 (fr) * 2015-07-21 2017-06-08 Indiana University Research & Technology Corporation Adn méthylé et déméthylé acellulaire dans les maladies résultant d'anomalies du taux de glucose dans le sang

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