WO1999043844A1 - Identification differentielle a soustraction reciproque - Google Patents

Identification differentielle a soustraction reciproque Download PDF

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Publication number
WO1999043844A1
WO1999043844A1 PCT/US1999/004323 US9904323W WO9943844A1 WO 1999043844 A1 WO1999043844 A1 WO 1999043844A1 US 9904323 W US9904323 W US 9904323W WO 9943844 A1 WO9943844 A1 WO 9943844A1
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nucleic acid
ell
isolated nucleic
pegen
samples
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PCT/US1999/004323
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English (en)
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Paul B. Fisher
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The Trustees Of Columbia University In The City Of New York
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Priority to AU28817/99A priority Critical patent/AU2881799A/en
Publication of WO1999043844A1 publication Critical patent/WO1999043844A1/fr
Priority to US09/644,460 priority patent/US6657053B1/en
Priority to US10/725,969 priority patent/US20040132076A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • Changes in gene expression are important determinants of normal cellular physiology, including cell cycle regulation, differentiation and development, and they directly contribute to abnormal cellular physiology, including developmental anomalies, aberrant programs of differentiation and cancer (1-4) .
  • the identification, cloning and characterization of differentially expressed genes will provide relevant and important insights into the molecular determinants of processes such as growth, development, aging, differentiation and cancer. A number of procedures can be used to identify and clone differentially expressed genes.
  • DDRT-PCR Since first introduced by Liang and Pardee (11) , DDRT-PCR has gained wide popularity in analyzing and cloning differentially expressed genes.
  • total RNAs or mRNAs from two or more cell types (or cells grown under different conditions, cells representing different stages of development, cells treated with agents modifying cellular physiology, etc.) are reverse-transcribed with two-base-pair anchored oligo dT primers, which divide mRNA populations into 12 cDNA subgroups. Then, each cDNA subgroup is amplified by PCR with one of 20 arbitrary 10-mer 5' primers and a 3' anchored primer and the PCR-amplified cDNA fragments are resolved in DNA sequencing gels.
  • the combinations of primers are designed not only to yield a detectable size and number of bands, but also to display nearly the complete repertoire of mRNA species.
  • DDRT-PCR is a powerful methodology in which a vast number of mRNA species (>20,000, if no redundancy occurs) can be analyzed with only a small quantity of RNA (about 5 ⁇ g) (11) .
  • DDRT-PCR is often the method of choice when the RNA source is limiting, such as tissue biopsies.
  • a direct advantage of DDRT-PCR is the ability to identify and isolate both up- and down-regulated differentially expressed genes in the same reaction.
  • the DDRT-PCR technique permits the display of multiple samples in the same gel, which is useful in defining specific diagnostic alterations in RNA species and for temporally analyzing gene expression changes.
  • the DDRT-PCR technique is not problem free.
  • Difficulties encountered when using standard DDRT-PCR include, a high incidence of false positives and redundant gene identification, poor reproducibility, biased gene display and lack of functional information about the cloned cDNA. Furthermore, poor separation can mask differentially expressed genes of low abundance under the intense signals generated by highly expressed genes. The - 3 - generation of false positives and redundancy can be highly problematic, resulting in an inordinate expenditure of resources to confirm appropriate differential expression and uniqueness of the isolated cDNAs .
  • the cDNAs must be isolated from the gels in pure form (contamination of bands with multiple sequences complicates clone identification) , reamplified, placed in an appropriate cloning vector, analyzed for authentic differential expression and finally sequenced.
  • Subtractive hybridization in which hybridization between tester and driver is followed by selective removal of common gene products, enriches for unique gene products in the tester cDNA population and reduces the abundance of common cDNAs (9) .
  • a subtracted cDNA library can be analyzed to identify and clone differentially expressed genes by randomly picking colonies or by differential screening (29-31) .
  • subtractive hybridization has been successfully used to clone a number of differentially expressed genes (5-7,10), this approach is both labor-intensive and does not result in isolation of the full spectrum of genes displaying altered expression
  • DDRT-PCR performed with subtracted RNA or cDNA samples represents a powerful strategy to clone up and down-regulated gene products.
  • This approach should result in the enrichment of unique sequences and a reduction or elimination of common sequences.
  • This scheme should also result in a consistent reduction in band complexity on a display gel, thereby permitting a clearer separation of cDNAs resulting in fewer false positive reactions.
  • it should be possible to use fewer primer sets for reverse transcription and PCR reactions to analyze the complete spectrum of differentially expressed genes.
  • rare gene products that are masked by strong common gene products should be displayed by using subtraction hybridization in combination with DDRT-PCR.
  • RSDD reciprocal subtraction differential RNA display
  • This invention provides a method for identifying differentially expressed nucleic acids between two samples, comprising: (a) selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids; (b) performing reciprocal subtraction between the nucleic acid samples to produce two subtracted nucleic acid samples; (c) amplifying the two subtracted nucleic acid samples; and (d) comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.
  • This invention also provides a method for identifying differentially expressed nucleic acids between two samples, comprising: (a) selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids; (b) amplifying the two nucleic acid samples; (c) performing reciprocal subtraction between the amplified nucleic acid samples to produce two subtracted nucleic acid samples; and (d) comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.
  • This invention further provides the above-described methods, wherein the first and second nucleic acid samples are obtained from cells in different developmental stages .
  • This invention further provides the above-described methods, wherein the first and second nucleic acid samples are obtained from cells from different tissue types .
  • this invention provides the above-described methods, wherein the 3' primer used in the PCR amplification is an oligo dT 3' primer. - 7 -
  • this invention provides the above-described methods, wherein the 3' primer used in the PCR amplification is a single anchor oligo dT 3' primer.
  • step (e) comprises using a gel to separate the nucleic acids from both of the libraries .
  • This invention provides the isolated nucleic acid identified by the above-described methods, wherein the nucleic acid was not previously known to be differentially expressed between the two samples.
  • RSDD differential RNA display pattern of conventional DDRT-PCR with RNA from Ell (C) and Ell-NMT
  • RSDD reciprocal subtraction differential RNA display
  • PEGen progression elevated genes
  • PSGen progression suppressed genes
  • RSDD reciprocal subtraction differential RNA display
  • reverse Northern blotting Dif ferential expression of representative progression elevated genes (PEGen) and progression suppressed genes (PSGen) identified by reciprocal subtraction differential RNA display (RSDD) and reverse Northern blotting .
  • PEGen progression elevated genes
  • PSGen progression suppressed genes
  • RSDD reciprocal subtraction differential RNA display
  • reverse Northern blotting Dif ferential expression of representative progression elevated genes (PEGen) and progression suppressed genes (PSGen) identified by reciprocal subtraction differential RNA display (RSDD) and reverse Northern blotting .
  • RNA display RSDD
  • reverse Northern blotting RSDD
  • Figure 5 cDNA fragment of PEGen 7 - 90% Homology to Human HPV16 E1BP. (Sequence ID No . 1)
  • Figure 8 cDNA fragment of PEGen 14. (Sequence ID No . 5)
  • Figure 12 cDNA fragment of PEGen 26 - Rat poly ADP-ribose polymerase. (Sequence ID No. 9)
  • Figure 18 cDNA fragment of PSGen 1 which has 80% homology to B . taurus supervillin. (Sequence ID No. 15)
  • Figure 20 cDNA fragment of PSGen 4 - Rat proteasome activator. (Sequence ID No. 17) - 11 -
  • Figure 21 cDNA fragment of PSGen 10 - Rat Ferritin Heavy Chain.
  • RSDD reciprocal differential RNA display
  • This invention provides a method for identifying differentially expressed nucleic acids between two samples, comprising: (a) selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids; (b) performing reciprocal subtraction between the nucleic acid samples to produce two subtracted nucleic acid samples; (c) amplifying the two subtracted nucleic acid samples; and (d) comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.
  • the nucleic acid samples are mRNA or derived from mRNA. In another embodiment, the nucleic acid samples are total RNA. In another embodiment, the nucleic acid samples are cDNA. In another embodiment, the nucleic acid samples are a nucleic acid library.
  • differentially expressed nucleic acids are expressed at different levels. In a further embodiment, one of the nucleic acids is not expressed. In a different embodiment, one of the nucleic acids is expressed in truncated form.
  • reciprocal subtraction includes using nucleic acid sample A to subtract common nucleic acids from nucleic acid sample B (based on hybridization) and also using nucleic acid sample B to subtract common nucleic acids from nucleic sample A.
  • the complement of nucleic acid sample A is used to subtract nucleic acids from nucleic acid sample B and the complement of nucleic acid sample B is used to subtract nucleic acids from nucleic acid sample A.
  • the RNA of nucleic acid sample A is used to subtract nucleic acids from nucleic acid sample B and the RNA of nucleic acid sample B is used to subtract nucleic acids from nucleic acid sample A.
  • the cDNA of nucleic acid sample A is used to subtract nucleic acids from nucleic acid sample B and the cDNA of nucleic acid sample B is used to subtract nucleic acids from nucleic acid sample A.
  • methods of amplification include PCR and rolling circle replication.
  • nucleic acid amplification A basic description of nucleic acid amplification is described in Mullis, U.S. Patent No. 4,683,202, which is incorporated herein by reference.
  • the amplification reaction uses a template nucleic acid contained in a sample, two primer sequences and inducing agents.
  • the extension product of one primer when hybridized to the second primer becomes a template for the production of a complementary extension product and vice versa, and the process is repeated as often as is necessary to produce a detectable amount of the sequence .
  • the inducing agent may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
  • Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, thermostable Taq DNA polymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase and other enzymes which will facilitate combination of the nucleotides in the proper manner to form amplification products.
  • the oligonucleotide primers can be synthesized by automated instruments sold by a variety of manufacturers or can be commercially prepared based upon the nucleic acid sequence of this invention.
  • This invention also provides a method for identifying differentially expressed nucleic acids between two samples, comprising: a) selecting a first and second nucleic acid sample; b) producing libraries for the first - 16 - and second nucleic acid sample; c) amplifying the two libraries; d) performing reciprocal subtraction between the amplified libraries to produce two subtracted libraries; and e) comparing the two subtracted libraries to identify differentially expressed nucleic acids.
  • This invention also provides a method for identifying differentially expressed nucleic acids between two samples, comprising: (a) selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids; (b) amplifying the two nucleic acid samples; (c) performing reciprocal subtraction between the amplified nucleic acid samples to produce two subtracted nucleic acid samples; and (d) comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.
  • This invention also provides the above-described methods, wherein the two subtracted nucleic acid samples from step c are amplified prior to the comparing of step d.
  • This invention also provides the above-described methods, wherein the each of the nucleic acid samples comprises a library of nucleic acids.
  • This invention also provides the above-described methods, wherein the nucleic acid samples are obtained from total cellular RNA purified by hybridization with oligo (dT) .
  • This invention also provides the above-described methods, wherein the nucleic acid samples are obtained from total RNA from Ell and Ell-NMT cells.
  • Ell is an adenovirus-transformed rat embryo cell line that acquires an aggressive oncogenic progression phenotype when injected into athymic nude mice and reisolated in cell culture (Ell-NMT) . - 17 -
  • This invention further provides the above-described methods, wherein the first and second nucleic acid samples are obtained from cells in different developmental stages .
  • This invention further provides the above-described methods, wherein the first and second nucleic acid samples are obtained from cells from different tissue types .
  • This invention further provides the above-described methods, wherein the first and second nucleic acid samples are obtained from cells that differ in their exposure to external factors or in their gene expression.
  • cells that differ in their exposure to external factors or in their gene expression includes any cells that may have different levels of gene expression, wherein some genes may not be expressed at all .
  • cells that differ in their exposure to external factors or in their gene expression includes any cells that are likely to have different levels of gene expression, wherein some genes may not be expressed at all.
  • cells that differ in their exposure to external factors or in their gene expression includes any cell that has a phenotypically recognizable difference.
  • a short list of examples of cells that differ in their exposure to external factors or in their gene expression includes: cancerous versus normal cells, advanced cancer progression cells versus ealier cancer stage cells, diseased cells versus nondiseased cells, infected cells versus noninfected cells, later developmental stage cells versus earlier developmental stage cells, cells after DNA damage versus cells before DNA damage, senescent cells versus younger cells, cells induced by growth factors versus cells not induced by growth factors, cells in the process of neurodegeneration versus normal cells, and cells exposed to a chemotherapeutic agent versus normal cells .
  • tissues types include but are not limited to tissues containing: cells grown under or exposed to different conditions, cells in different stages of development, cells treated with agents modifying cellular physiology, and cells having different functions .
  • cells at different stages of development are cells taken or analyzed at times differing by one or more hours in the development of the cell or organism.
  • step (d) comprises PCR amplification.
  • this invention provides the above-described methods, wherein the 3' primer used in the PCR amplification is an oligo dT 3' primer.
  • the 3' primer used in the PCR amplification is an oligo dT 3' primer.
  • oligo dT primers are T 13 , T 13 A, and T 13 GA.
  • this invention provides the above-described methods, wherein the 3' primer used in the PCR amplification is a single anchor oligo dT 3' primer.
  • Olgio dT 3' primers include T 13 A, T 13 C, and T 13 G.
  • This invention provides the above-described methods, wherein the PCR amplification uses a set of random primers .
  • This invention provides the above-described methods, wherein the 5' primer is an arbitrary primer. - 19 -
  • step (e) comprises using a gel to separate the nucleic acids from both of the substracted libraries.
  • the gel is a polyacrylamide gel. In another embodiment, the gel is an agarose gel.
  • This invention further provides the above-described methods, further comprising PCR amplifying the first and second nucleic acid samples.
  • This invention also provides the above-described methods, further comprising reamplifying differentially expressed bands.
  • This invention also provides the above-described methods, further comprising reamplifying differentially expressed nucleic acid.
  • differentially amplified bands from plasmids of each subtracted library were marked with an 18G needle through the film and cut out with a razor.
  • the cut out differentially expressed bands can be reamplified (i.e. by PCR) and examined by reverse Northern and Northern blot analyses.
  • this invention provides the above-described methods, wherein the comparing of step (e) comprises comparing the band intensities of the two amplified differentially expressed nucleic acids.
  • this invention provides the above-described methods, wherein the nucleic acid samples are mRNA or cDNA derived from mRNA. - 20 -
  • this invention provides the above-described methods, wherein the comparing of step (e) comprises comparing the quantities of the two amplified differentially expressed nucleic acids.
  • This invention further provides the above-described methods, wherein the differences in band intensity between the two subtracted libraries are electronically quantified.
  • This invention further provides the above-described methods, wherein the differences in the quantities of nucleic acid between the two subtracted libraries are electronically quantified.
  • electronic quantification involves using a scanner to detect the bands.
  • computer software such as Corel Draw, can be used to determine the pixel intensity of the scanned image, thereby quantifying the band intensity.
  • this invention provides the above-described methods, wherein the libraries of step (b) are constructed with ⁇ -ZAP cDNA library kits.
  • the libraries of step (b) are constructed with ⁇ -ZAP cDNA library kits.
  • any cDNA library would be suitable .
  • This invention provides the isolated nucleic acid identified by the above-described methods, wherein the nucleic acid was not previously known.
  • This invention also provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 12 (Al 144569) .
  • this invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid - 21 - is the nucleic acid designated PSGen 13 (Accession No. Al 144570) .
  • This invention provides the above -described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 23.
  • This invention provides the above -described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 24.
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 25.
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 26 (Accession No. Al 144571) .
  • This invention also provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 27 (Accession No. Al 144572) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 28 (Al 144573) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PSGen 29 (Al 144574) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 13 (Al 144564) . - 22 -
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 14 (Al 144565) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 15.
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 24 (Accession No. Al 144566) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 28 (Al 144567) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 32 (Al 144568) .
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 42.
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 43.
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 44.
  • This invention provides the above-described isolated nucleic acid, wherein the isolated nucleic acid is the nucleic acid designated PEGen 48. - 23 -
  • This invention further provides a previously unknown isolated nucleic acid molecule identified by the above- described methods which comprises (a) one of the nucleic acid sequences as set forth in Figure 35; (b) a sequence being degenerated to a sequence of (a) as a result of the genetic code; (c) a sequence encoding one of the amino acid sequences as set forth in Figure 35. (d) a sequence of at least 12 nucleotides capable of specifically hybridizing to the sequence of (a) , (b) or (c) .
  • this invention provides a purified polypeptide comprising one of the amino acid sequence as set forth in Figure 35.
  • RSDD reciprocal subtraction differential RNA display
  • the model used for RSDD was an adenovirus-transformed rat embryo cell line, Ell, that acquires an aggressive oncogenic progression phenotype when injected into athymic nude mice and reisolated in cell culture (Ell-NMT) (10,33,34).
  • Ell-NMT cell culture
  • Injection of Ell cells into nude mice results in tumors in 100% of animals with a tumor latency time of approximately 35 to 40 days
  • Ell-NMT cells form tumors in 100% of nude mice with a tumor latency time of 15 to 20 days (10,34,35) .
  • Ell cells form colonies in agar with an efficiency of ⁇ 3%
  • Ell-NMT display an agar cloning efficiency of >30% (10,33,34).
  • the increased tumorigenicity and enhanced anchorage independence phenotypes are key indicators of tumor progression in the E11/E11-NMT model system (10,33,34).
  • RNA display was directly performed with reciprocally subtracted cDNA plasmid libraries (Ell minus Ell-NMT and Ell-NMT minus Ell) .
  • the subtracted cDNA libraries used in this experiment are free from potential PCR artifacts and provide more stable and consistent sources for DDRT-PCR analyzes.
  • three single anchored oligo dT 3 ' primers were used instead of two-base-anchored approach described by Hakvoort et al (32) .
  • reamplified cDNAs identified using RSDD were analyzed using the reverse Northern blotting procedure
  • RNA from Ell and Ell-NMT cells was isolated by the guanidinium isothiocyanate/CsCl centrifugation procedure and poly A + RNA was purified with oligo (dT) cellulose chromatography (5) .
  • Two ⁇ -ZAP cDNA libraries from Ell and Ell-NMT mRNA's were constructed with ⁇ -ZAP cDNA library Kits (Stratagene) following the manufacturer's protocol. Reciprocal subtraction between Ell and Ell-NMT libraries was performed and two subtracted cDNA libraries (Ell minus Ell-NMT and Ell-NMT minus Ell) were constructed as described previously.
  • Bacterial plasmid libraries from the subtracted ⁇ -ZAP cDNA libraries were obtained by in vi vo excision following the manufacturer's protocol (Stratagene) and the plasmids were isolated with Qiagen columns (Qiagen Inc.) .
  • the purified plasmids of reciprocally subtracted cDNA libraries were directly subjected to differential display as in Liang et . al . (38) with minor modifications.
  • the plasmids of reciprocally subtracted cDNA libraries were PCR-amplified with the combination of three single-anchor 3' primers (T 13 A, T 13 C or T 13 G) and 18 arbitrary 5' 10-mer primers obtained from Operon Technology Inc. (Alameda, CA. OPA 1-20 except 0PA1 and 3) .
  • the 20 ⁇ l PCR reaction consisted of 10 mM Tris-HCl pH 8.4 , 50 mM KCl , 1.5 mM MgCl 2 , 2 ⁇ M each dNTP, 0.2 ⁇ M 5' arbitrary primer, 1 ⁇ M 3 ' anchor primer, 50 ng of plasmid of a subtracted library, 10 ⁇ Ci - 35 S-dATP (3000 Ci/mmole from Amersham) and 1 U of Taq DNA polymerase (Gibco BRL) .
  • the parameters of PCR were 30 sec at 95 C, 40 cycle of 30 sec at 95 C, 2 min. at 40 C and 30 sec at 72 C and additional 5 min. at 72 C.
  • the supernatant was collected and stored at -20 °C until reamplification.
  • the band extract was reamplified with the same cycling parameters in a 50 ⁇ l reaction consisting of 10 mM Tris-HCl pH 8.4 , 50 mM KC1, 1.5 mM MgCl 2 , 20 ⁇ M each dNTP, 0.2 ⁇ M 5' arbitrary primer, 1 ⁇ M 3' anchor primer, 5 ⁇ l of band extract and 2.5 U of Taq DNA polymerase (Gibco BRL) .
  • RNA from Ell and Ell-NMT cells were run side-by-side in a 1% agarose gel with formaldehyde and transferred to a positively charged Nylon membrane.
  • Reamplification reaction (5 ⁇ l) was 32 P-labeled with a multiprime labeling kit (Boehringer
  • RNAs from various Ell and Ell-NMT derivatives displaying either a progressed or suppressed progression phenotype, based on nude mice tumorigenesis and soft agar cloning assays were analyzed.
  • Subtraction hybridization provides a direct means of enriching for unique cDNA species and eliminating common sequences between complex genomes.
  • DDRT-PCR is a proven methodology for the rapid identification and cloning of differentially expressed sequences between cell types
  • Hakvoort et . al . (32) used a reciprocal subtraction approach to analyze gene expression changes resulting during liver regeneration following 70% hepatectomy, i.e., normal liver subtracted from partially hepatectomized regenerating liver and vice versa. Although some bands displayed apparent enrichment, the complexity of the display pattern did not show appreciable simplification. These results are in stark contrast to RSDD, which results in a clear delineation and simplification of differentially expressed amplified bands (Figs. 1) . Although conceptually similar, RSDD is significantly more effective than the subtraction plus DDRT-PCR approach described by Hakvoort et al . (32) . The improved efficiency of RSDD versus the Hakvoort et al .
  • Hakvoort et al . (32) approach can be attributed to several factors.
  • the approach of Hakvoort et al . (32) is based on the subtraction procedure described by Wang and Brown (38) . This approach involves multiple rounds of PCR-amplification prior to each round of subtractive hybridization.
  • RSDD involves a single round of reciprocal subtraction that does not involve PCR amplification (5,10) .
  • the complicated display pattern observed by Hakvoort et al . (32) even after three or four rounds of subtraction might result from reduced subtraction efficiency, PCR artifacts or a combination of these problems.
  • Increasing the number of reactions by using two-base pair anchored oligo dT primers did not reduce the complexity of displayed bands - 30 -
  • a critical component for the successful use of RSDD involves the use of an appropriate subtraction hybridization protocol, that can efficiently reduce cDNA complexity and generate stable populations of cDNAs for analysis .
  • DDRT-PCR can generate large numbers of di ferentially displayed bands making subsequent analysis both labor intensive and a daunting challenge.
  • RSDD has been used in combination with reverse Northern analyses of isolated cDNAs .
  • Gel extracted cDNA fragments were reamplified, dot-blotted on Nylon membranes and successively probed with reverse transcribed 32 P-cDNA from Ell or Ell-NMT RNAs
  • the signal intensities of the various cDNAs in reverse Northern analysis were quantified and normalized against that of GAPDH, which remained unchanged in Ell and Ell-NMT cells.
  • the PEG-3 (PEGen-3) gene (10) was used as an additional control, to verify increased expression in Ell-NMT versus Ell cells.
  • PEGen-3 levels were 4-fold higher in Ell-NMT than in Ell cells, which coincided with Northern blotting results, thereby demonstrating the concordance of reverse Northern and Northern assays.
  • a > 1.8-fold differential cut-off (after normalization for GAPDH expression) was used to identify and isolate cDNA bands displaying modified expression in Ell versus Ell-NMT cells.
  • SSCP single strand conformational polymorphism
  • reverse Northern analyses using cloned cDNA populations (39,40) .
  • Ell-NMT ( ⁇ 80% concordance) or a larger panel of cells differentially displaying the progression phenotype, including progression negative, Ell, CREF x Ell-NMT FI, CREF X Ell-NMT F2 , Ell X Ell-NMT A6 , Ell X Ell-NMT 3b, Ell-NMT Aza Bl and Ell-NMT Aza CI , and progression positive Ell-NMT, CREF X Ell-NMT Rl , CREF X Ell-NMT R2 , Ell X Ell-NMT A6TD, Ell X Ell-NMT Ila, Ell-ras and Ell-HPV E6/E7.
  • PEGen progression upregulated genes
  • PSGen progression suppressed genes
  • PEGen 7 is expressed at ⁇ 5-fold higher levels in Ell-NMT than in Ell cells.
  • PEGen 7 is ⁇ 90% homologous to 16E1-BP, a cDNA encoding a protein identified using the yeast two-hybrid assay that interacts with human papillomavirus type 16 El protein (41) .
  • 16E1-BP encodes a 432aa protein of unknown function but does contain an ATPase signature motif (Gly-X 4 -Gly consensus ATP binding motif at aa 179 through 186) .
  • 16E1-BP appears to be a form of TRIP13, a protein previously shown to bind thyroid hormone receptor in yeast two-hybrid assays.
  • the role of PEGen 7/16E1-BP in the progression phenotype in the E11/E11-NMT progression model is not known. Additional studies are necessary to determine if this gene change is associative or causative of transformation progression.
  • PEGen 8 is expressed at ⁇ 3- to 4- fold higher levels in Ell-NMT than in Ell cells.
  • PEGen 8 shows 100% homology to rat phosphofructokinase C (PFK-C) (42) .
  • PFK catalyzes the rate-limiting and committed step in glycolysis, the conversion of fructose 6 -phosphate to fructose 1, 6-biphosphate.
  • Three subunit isozymes of PFK have been identified, that form homo- and heterotetramers with differing catalytic and allosteric properties.
  • PFK-M is - 37 - specific for cardiac and skeletal muscle
  • PFK-L is expressed in many tissues but is most abundant in the liver
  • PFK-C is expressed in several brain regions and the anterior pituitary but not in liver, skeletal muscle, or several other human tissues.
  • the cDNA of PFK-C isolated from a rat hypothalamic cDNA library is 2643 bp and encodes a protein of 765aa (42) .
  • Sanchez-Martinez and Aragon (43) demonstrated that PFK-C is the predominant form of PFK in ascites tumor cells (obtained from a transplantable mouse carcinoma of mammary origin) , whereas PFK-L is most abundant in the normal mammary gland.
  • PEGen 21 is expressed at ⁇ 3- to 4-fold higher levels in Ell-NMT than in Ell cells. PEGen 21 displays ⁇ 94% homology with the fibroblast growth factor-4 inducible gene FIN-14 (44) .
  • FIN-14 is a novel cDNA of unknown function that hybridizes with a 4.5 kb mRNA that is induced 4 -fold in NIH3T3 mouse cells following treatment with FGF-4. The induction of FIN-14 occurs late (18 hr) after treatment with FGF-4 and does not occur when cells are treated for 18 hr with FGF-4 in the presence of cycloheximide (44) . These results confirm that FIN-14 encodes a late-inducible gene.
  • FIN-14 is trancriptionally activated in NIH3T3 cells following growth factor stimulation.
  • Tissue distribution studies indicate expression of a single mRNA species in the kidney with low levels of expression observed in several other tissues including testis and thymus .
  • Mouse embryogenesis studies indicate that FIN-14 expression occurs constitutively in mouse embryos between day 10.5 and 15.5.
  • FIN-14 was constitutively expressed in PC12 cells and its level - 38 - did not vary appreciably in response to growth factor stimulation.
  • the role of PEGen 21/FIN-14 in progression in Ell/Ell-NMT model system is not currently known.
  • PSGen cDNAs PSGen-12 and PSGen- 13, consist of sequences without homology to those presently reported in various DNA databases. Expression of these cDNAs is ⁇ 3- to 4-fold higher in Ell versus Ell-NMT cells (Fig. 3) . It is not currently known whether these genes simply correlate with or functionally regulate the progression phenotype.
  • the identification of full-length cDNAs for PSGen-12 and PSGen-13 are in progress and once identified experiments can be conducted to directly define the role of these PSGen ' s in cancer progression.
  • RSDD represents a method of choice either as a more efficient and less time consuming modification of the differential RNA display strategy or as a screening methodology for identifying differentially expressed genes in reciprocally subtracted cDNA libraries.
  • RSDD adenovirus-transformed rat embryo cell line
  • Ell an adenovirus-transformed rat embryo cell line
  • Ell-NMT cell culture
  • Ell cells form colonies in agar with an efficiency of ⁇ 3 %, whereas Ell-NMT display an agar cloning efficiency of >30% (6,26,27).
  • the increased tumorigenicity and enhanced anchorage independence phenotypes are key indicators of tumor progression in the Ell/Ell-NMT model system
  • RSDD has resulted in the identification and cloning of genes displaying elevated expression in progressed tumor cells (progression elevated gene, PEGen) and suppressed expression in progressed tumor cells (progression suppressed gene, PSGen) .
  • RNA isolation and cDNA library construction Total RNA from Ell and Ell-NMT cells was isolated by the guanidinium isothiocyanate/CsCl centrifugation procedure and poly (A) + RNA was purified with oligo (dT) cellulose chromatography (5) .
  • Two ⁇ -ZAP cDNA libraries from Ell and Ell-NMT mRNAs were constructed with ⁇ -ZAP cDNA library kits (Stratagene) following the manufacturer's protocol. Reciprocal subtraction between Ell and Ell-NMT libraries was performed and two subtracted cDNA libraries (Ell minus Ell-NMT and Ell-NMT minus Ell) were constructed as - 48 - described (5, 6) .
  • Plasmid cDNA libraries from the subtracted ⁇ -ZAP cDNA libraries were obtained by in vivo excision following the manufacturer's protocol (Stratagene) and the plasmids were isolated with Qiagen columns (Qiagen, Chatsworth, CA. ) .
  • the purified plasmids of reciprocally subtracted cDNA libraries were directly subjected to differential display as in Liang et al . (28) with minor modifications.
  • the plasmids of reciprocally subtracted cDNA libraries were PCR-amplified with the combination of three single-anchor 3 ' primers (T 13 A, T 13 C or T 13 G) and 18 arbitrary 5' 10-mer primers obtained from Operon Technology Inc. (Alameda, CA. OPA 1-20 except OPAl and 3) .
  • the 20 ⁇ l PCR reaction consisted of 10 mM Tris-HCl
  • DNA sequencing gel maintained at 50°C. PCR reactions of plasmids from each subtracted library in a primer set were run side by side. Differentially amplified bands from plasmids of each subtracted library were marked with 18G needle through the film and cut out with a razor. The gel slice was put in 100 ⁇ l TE (pH 8.0) and incubated at 4°C overnight. After the incubation, the mixture was boiled for 5 min and microcentrifuged for two min. The supernatant was collected and stored at -20°C until reamplification. The band extract was reamplified with the same cycling parameters in a 50 ⁇ l reaction consisting of 10 mM Tris-HCl (pH 8.4), 50 mM KCl , 1.5 mM - 49 -
  • RNA from Ell and Ell-NMT cells were run side-by-side in a 1% agarose gel with formaldehyde and transferred to a positively charged Nylon membrane.
  • Reamplification reaction (5 ⁇ l) was 32 P-labeled with a multiprime labeling kit (Boehringer Mannheim) used to probe the membrane as described above.
  • DNA fragments expressed differentially between Ell and Ell-NMT in Northern blot analyses were cloned into the EcoRV site of the pZEro-2.1 cloning vector (Invitrogene) and sequenced.
  • RNAs from various Ell and Ell-NMT derivatives displaying either a progressed or suppressed progression phenotype, based on nude mice tumorigenesis and soft agar cloning assays were analyzed.
  • Subtraction hybridization provides a direct means of enriching for unique cDNA species and eliminating common sequences between complex genomes (7, 18) .
  • DDRT-PCR is a proven methodology for the rapid identification and cloning of differentially expressed sequences between cell types (1,2,28) .
  • subtraction hybridization combined with DDRT-PCR should reduce band complexity which often obscures the identification of differentially expressed genes and generates false positive signals (21,29) .
  • RSDD has been used to analyze genes differentially expressed during transformation progression (Fig. 28) .
  • Differential RNA display was directly performed with reciprocally subtracted cDNA plasmid libraries (Ell minus Ell-NMT and Ell-NMT minus Ell) that had not been subjected to PCR.
  • cDNAs identified using RSDD were analyzed using the reverse Northern blotting procedure (30,31). cDNAs displaying differential expression by reverse Northern blotting were subsequently confirmed for true differential expression by Northern analysis .
  • RNA display pattern of Ell and Ell-NMT cells using standard differential RNA display (DDRT-PCR) and RSDD is shown in Fig. 1 (Left Panel) .
  • the differential RNA display pattern of RSDD is much less complex than that of DDRT-PCR.
  • Hakvoort et al.(25) used a reciprocal subtraction approach to analyze gene expression changes resulting during liver regeneration following 70% hepatectomy, i.e., normal liver subtracted from partially hepatectomized regenerating liver and vice versa. Although some bands displayed apparent enrichment, the complexity of the display pattern did not show appreciable simplification. In contrast, RSDD results in a clearer delineation and simplification of differentially expressed amplified bands (Figs. 1) . Although conceptually similar, RSDD is significantly more effective than the subtraction plus DDRT-PCR approach described by Hakvoort et al . (25) The reasons for the improved efficiency of RSDD versus the Hakvoort et al . (25) approach are not known.
  • Hakvoort et al . (25) is based on the subtraction procedure described by Wang and Brown (32) . This approach uses multiple rounds of PCR-amplification prior to each round of subtractive hybridization.
  • RSDD involves a single round of reciprocal subtraction without intermediate amplification (5, 6) .
  • the complicated display pattern observed by Hakvoort et al . (25) even after three or four rounds of subtraction might result from reduced subtraction efficiency, PCR artifacts or a combination of these problems.
  • a critical component for the successful use of RSDD involves the use of an appropriate subtraction hybridization protocol, which can efficiently reduce cDNA complexity and generate stable populations of cDNAs for analysis .
  • DDRT-PCR can generate large numbers of differentially displayed bands making subsequent analysis both labor intensive and a daunting challenge.
  • RSDD has been used in combination with reverse Northern analyses of isolated cDNAs .
  • Gel extracted cDNA fragments were reamplified, dot-blotted on Nylon membranes and successively probed with reverse transcribed 32 P-cDNA from Ell or Ell-NMT RNAs - 54 -
  • Ell-NMT cells correlates with increased expression of a large number of genes, whereas only a smaller subset of genes display decreased expression.
  • SSCP single strand conformational polymorphism
  • Figs. 29 and 30 The expression pattern of representative RSDD-derived cDNAs in Ell versus Ell-NMT and in a more expanded Ell/Ell-NMT progression cell culture series is shown in Figs. 29 and 30, respectively.
  • Reverse Northern results correlated well with Northern blots using Ell and Ell-NMT ( ⁇ 75% concordance) or a larger panel of cells differentially displaying the progression phenotype, including progression negative Ell, CREF x Ell-NMT FI and F2, Ell x Ell-NMT A6 , Ell x Ell-NMT 3b, Ell-NMT Aza Bl and Aza CI cells, and progression positive Ell-NMT, CREF x Ell-NMT Rl and R2 , Ell x Ell-NMT A6TD, Ell x Ell-NMT
  • Novel PEGen cDNAs include, PEGen 13, 14, 24, 28 and 32.
  • Known PEGen genes included PEGen 7 (human papilloma virus-16 early region 1 binding protein; HPV16 E1BP) , PEGen 8 (phosphofructokinase kinase C; PFK-C) , PEGen 21 (a fibroblast growth factor-4 inducible gene; FIN 14) , PEGen 26 (poly ADP-ribose polymerase) and PEGen 30 (rat espl homology) .
  • PSGen cDNAs six of six (100%) were novel, including PSGen 12, 13, 26, 27, 28 and 29 (Table 3) .
  • PEGen progression elevated genes that display elevated expression in Ell-NMT versus Ell cells.
  • PSGen progression suppressed genes that display elevated expression in Ell versus Ell-NMT cells.
  • ""Sequences have compared with reported genes in various
  • DNA data bases including GenBank and EMBL
  • GenBank GenBank and EMBL
  • Genes without homology to currently reported genes are indicated as unknown.
  • percentage homology with known sequences, either human, rat or mouse is indicated.
  • PEGen 7 is expressed at ⁇ 4-fold higher levels in Ell-NMT than in Ell cells.
  • PEGen 7 is ⁇ 98% homologous to 16E1-BP, a cDNA encoding a protein identified using the yeast two-hybrid assay that interacts with human papillomavirus type 16 El protein (35) .
  • 16E1-BP encodes a 432aa protein of unknown function but does contain an ATPase signature motif (Gly-X4-Gly consensus ATP binding motif at aa 179 through 186).
  • 16E1-BP appears to be a form of TRIP13, a protein previously shown to bind thyroid hormone receptor in yeast two-hybrid assays.
  • the role of PEGen 7/16E1-BP in the progression phenotype in the Ell/Ell-NMT progression model is not known. Additional studies are necessary to determine if this gene change is associative or causative of transformation progression.
  • PEGen 8 is expressed at ⁇ 3- to 4- fold higher levels in Ell-NMT than in Ell cells.
  • PEGen 8 shows 100% homology to rat phosphofructokinase C (PFK-C) (36) .
  • PFK catalyzes the rate-limiting and committed step in glycolysis, the conversion of fructose 6-phosphate to fructose 1, 6-biphosphate .
  • Three subunit isozymes of PFK have been identified, that form homo- and heterotetramers with differing catalytic and allosteric properties.
  • PFK-M is specific for cardiac and skeletal muscle
  • PFK-L is expressed in many, tissues but is most abundant in the liver
  • PFK-C is expressed in several brain regions and the anterior pituitary but not in liver, skeletal muscle, or several other human tissues.
  • the cDNA of PFK-C isolated from a rat hypothalamic cDNA library is 2643 bp and encodes a protein of 765aa (-36) .
  • Sanchez-Martinez and Aragon (37) demonstrated that PFK-C is the predominant form of PFK in ascites tumor cells (obtained from a transplantable mouse carcinoma of mammary origin) , whereas PFK-L is most abundant in the normal mammary gland.
  • PEGen 21 is expressed at ⁇ 3- to 4-fold higher levels in Ell-NMT than in Ell cells. PEGen 21 displays ⁇ 98% homology with the fibroblast growth factor-4 inducible gene FIN- 14 (38) .
  • FIN-14 is a novel cDNA of unknown function that hybridizes with a 4.5 kb mRNA that is induced 4-fold in NIH 3T3 mouse cells following treatment with FGF-4. The induction of FIN-14 occurs late (18 hr) after treatment with FGF-4 and does not occur when cells are treated for 18 hr with FGF-4 in the presence of cycloheximide (38) . These results confirm that FIN-14 encodes a late- inducible gene.
  • FIN-14 is transcriptionally activated in NIH 3T3 cells following growth factor stimulation.
  • Tissue distribution studies indicate expression of a single mRNA species in the kidney with low levels of expression observed in several other tissues including testis and thymus .
  • Mouse embryogenesis studies indicate that FIN-14 expression occurs constitutively in mouse embryos between day 10.5 and 15.5.
  • FIN-14 was constitutively expressed in PC12 cells and its level did not vary appreciably in response to growth factor stimulation.
  • the role of PEGen 21/FIN-14 in progression in Ell/Ell-NMT model system is not currently known.
  • PEGen 26 is expressed at ⁇ 3- to 4-fold higher levels in Ell-NMT than in Ell cells.
  • This cDNA is identical to rat poly (ADP-ribose) polymerase (PARP) (39) .
  • PARP contributes to the ability of eukaryotic cells to contend with both environmental and endogenous genotoxic agents (40) .
  • PARP is a nuclear enzyme that binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly (ADP-ribose) (39,40).
  • PARP activity contributes to the recovery of proliferating cells from DNA damage - 59 - and to the maintenance of genomic stability, which may be regulated by effects on chromatin structure, DNA base-excision repair and cell cycle regulation (39,40).
  • PEGen 26/PARP The role of PEGen 26/PARP in mediating the progression phenotype is not currently known. However, since cancer is a progressive disease characterized by the accumulation of genetic alterations in the evolving tumor (6), it is believed that overexpression of PEGen 26/PARP in Ell-NMT may facilitate the ability of these aggressive cancer cells to maintain genomic stability during cancer progression. In this context, PEGen 26/PARP may be an integral component of progression. This hypothesis is readily testable. PEGen 30 is expressed at 2- to 3-fold higher levels in Ell-NMT than in Ell cells. This cDNA displays ⁇ 98.5% homology to rat espl (41) .
  • Rat espl encodes a 24 -kDa nuclear protein which is the rat homologue of Drosophila Enhancer of split., a gene involved in ventral ectodermal development in Drosophila (41) .
  • PEGen 30 appears to be a homologue of espl, since the message detected in Ell and Ell-NMT cells
  • PSGen cDNAs 12, 13, 26, 27, 28 and 29, consist of sequences without homology to those in various DNA data bases. Expression of PSGen 12 and PSGen 13 cDNAs is ⁇ 3- to 4-fold higher in Ell versus Ell-NMT cells (Fig. 29) . It is not currently known whether these genes simply correlate with or functionally regulate the progression phenotype.
  • the identification of full-length cDNAs for PSGen- 12 and PSGen-13, as well as the other novel PSGen and PEGen cDNAs, are in progress and once isolated experiments can be conducted to directly define the role of these progression-related genes in cancer progression. - 60 -
  • RSDD modified gene- identification and gene-cloning technique
  • RSDD represents a method of choice either as a more efficient and less time consuming modification of the differential RNA display strategy or as a screening methodology for identifying differentially expressed genes in reciprocally subtracted cDNA libraries .
  • the ability of RSDD to identify differentially expressed genes that are dissimilar to those recognized using standard DDRT-PCR or subtraction hybridization indicates that this approach will be a valuable adjunct in cloning the complete repertoire of differentially expressed gene changes occurring between complex genomes .

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Abstract

L'invention concerne un procédé pour l'identification d'acides nucléiques exprimés différentiellement entre deux échantillons, caractérisé en ce qu'il comprend les étapes ci-après: a) sélection d'un premier et d'un deuxième échantillons d'acide nucléique; b) production de bibliothèques pour le premier et le deuxième échantillons d'acide nucléique; c) effectuer la soustraction réciproque entre les bibliothèques, de manière à produire deux bibliothèques soustraites; d) amplification des deux bibliothèques soustraites; et e) comparer les deux bibliothèques soustraites amplifiées en vue d'identifier des acides nucléiques exprimés différentiellement. L'invention concerne en outre le procédé précité où l'amorce 3' utilisée dans l'amplification PCR est une amorce 3' oligo dT. L'invention concerne en outre les procédés précités où la comparaison de l'étape e) comprend l'utilisation d'un gel pour séparer les acides nucléiques des deux bibliothèques. L'invention concerne également l'acide nucléique isolé, identifié par les procédés précités, où l'acide nucléique n'était pas antérieurement connu comme étant différentiellement exprimé entre les deux échantillons.
PCT/US1999/004323 1998-02-27 1999-02-26 Identification differentielle a soustraction reciproque WO1999043844A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023419A2 (fr) * 1999-09-27 2001-04-05 Scios Inc. Facteurs secretes
WO2002014360A1 (fr) * 2000-08-16 2002-02-21 Acologix, Inc Motif de fixation a integrine contenant des peptides et procedes servant a traiter des maladies du squelette
WO2002016419A2 (fr) * 2000-08-25 2002-02-28 The Trustees Of Columbia University In The City Of New York Gene 13 (psgen 13) a progression supprimee et ses utilisations
US7271151B2 (en) 1998-05-18 2007-09-18 Acologix, Inc. Polypeptide hormone phosphatonin
US7498021B2 (en) 2000-08-16 2009-03-03 Acologix, Inc. Dental products comprising bone growth enhancing peptide
US7622438B1 (en) 2005-07-18 2009-11-24 Acologix, Inc. Protein formulation for promoting hard tissue formation
US7888462B2 (en) 2007-01-22 2011-02-15 Acologix, Inc. Peptide composition and a method of promoting cartilage formation
US8053232B2 (en) 2004-01-23 2011-11-08 Virxsys Corporation Correction of alpha-1-antitrypsin genetic defects using spliceosome mediated RNA trans splicing

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981783A (en) * 1986-04-16 1991-01-01 Montefiore Medical Center Method for detecting pathological conditions
US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981783A (en) * 1986-04-16 1991-01-01 Montefiore Medical Center Method for detecting pathological conditions
US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7271151B2 (en) 1998-05-18 2007-09-18 Acologix, Inc. Polypeptide hormone phosphatonin
US7473771B2 (en) 1998-05-18 2009-01-06 University College London Polypeptide hormone phosphatonin
US7459300B2 (en) 1998-05-18 2008-12-02 University College London Polypeptide Hormone Phosphatonin
WO2001023419A3 (fr) * 1999-09-27 2001-08-30 Scios Inc Facteurs secretes
WO2001023419A2 (fr) * 1999-09-27 2001-04-05 Scios Inc. Facteurs secretes
US7498021B2 (en) 2000-08-16 2009-03-03 Acologix, Inc. Dental products comprising bone growth enhancing peptide
US7160862B2 (en) 2000-08-16 2007-01-09 Acologix, Inc. Integrin binding motif containing peptides and methods of treating skeletal diseases
WO2002014360A1 (fr) * 2000-08-16 2002-02-21 Acologix, Inc Motif de fixation a integrine contenant des peptides et procedes servant a traiter des maladies du squelette
WO2002016419A3 (fr) * 2000-08-25 2002-10-10 Univ Columbia Gene 13 (psgen 13) a progression supprimee et ses utilisations
WO2002016419A2 (fr) * 2000-08-25 2002-02-28 The Trustees Of Columbia University In The City Of New York Gene 13 (psgen 13) a progression supprimee et ses utilisations
US8053232B2 (en) 2004-01-23 2011-11-08 Virxsys Corporation Correction of alpha-1-antitrypsin genetic defects using spliceosome mediated RNA trans splicing
US7622438B1 (en) 2005-07-18 2009-11-24 Acologix, Inc. Protein formulation for promoting hard tissue formation
US7638486B2 (en) 2005-07-18 2009-12-29 Acologix, Inc. Method for promoting hard tissue formation
US7888462B2 (en) 2007-01-22 2011-02-15 Acologix, Inc. Peptide composition and a method of promoting cartilage formation
US8426558B2 (en) 2007-01-22 2013-04-23 Orthotrophix, Inc. Peptide composition and a method of promoting cartilage formation

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