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Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents

A technology of recombinant nucleic acid and pluripotent stem cells, which is applied in the field of pluripotent cells that use inducible recombinant ribonucleic acid factors to generate tumor-free embryonic stem cells, and can solve the problems of undetermined overall developmental signals

Active Publication Date: 2013-08-21
林希龙 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the mechanism of iPS cell formation is still unclear, this technology requires multiple transcriptional regulators to coordinate the switch between reactivation of embryonic genes and inactivation of developmental signals
Combinatorial effects of the Oct4-Sox2-c-Myc-Klf4 or Oct4-Sox2-Nanog-LIN28 genes appear to directly activate certain embryonic genes; however, how these effects result in the abrogation of the overall developmental signal necessary for somatic differentiation has not been determined

Method used

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  • Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents
  • Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents
  • Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents

Examples

Experimental program
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Effect test

Embodiment 2

[0046] The present inventors took advantage of the intracellular spliceosome, exosome and NMD system to catalyze the release of intronic mir-302 miRNA / shRNA factors from the SpRNAi-RGFP transgene. In Example 1 and Figure 2A and Figure 2B The sequence DNA recombination [for example: snRNPs U1, U2 and U4 / U6. U5tri-snRNP required by the sequence DNA recombination of the splice body components in the several snRNP recognition positions of the SpRNAi is described in it, and it comprises: 5' end splice site (SEQ.ID.NO.4), branch point motif (BrP; SEQ.ID.NO.6), polypyrimidine string (PPT; SEQ.ID.NO.7 or SEQ.ID.NO .8), and the 3' end splice site (SEQ.ID.NO.5)], used to assemble the synthetic snRNP recognition element into the SpRNAi intron and incorporate this artificially recombined SpRNAi into the isolated inducible RGFP gene To form the method for the SpRNAi-RGFP transgene. In addition, the SpRNAi further comprises an intron insertion site located between the 5' end splice sit...

Embodiment 1

[0147] Constructs comprising the SpRNAi recombinant RGFP gene (SpRNAi-RGFP)

[0148] Synthetic oligonucleotides used to generate SpRNAi introns containing sense, antisense, or hairpin EGFP inserts are listed below: sense N1,5'-GTAAGAGGATCCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3' (SEQ.ID.NO. 14); antisense N1, 5'-CGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGG-ATCC TCTTAC-3' (SEQ.ID.NO.15); sense N2, 5'-GTAAGAGGATCCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3' (SEQ.ID.NO. 16); antisense N2, 5'-CGCGTCAGGA GCGCACCATC TTCTTCAAGCGATCGGATCC TCTTAC-3' (SEQ.ID.NO.17); sense N3, 5'-GTAAGAGGATCCGATCGCAG GAGCGCACCA TCTTCTTCAA GTTAACTTGAAGAAGATGGT GCGCTCCTGA-3' (SEQ.ID.NO.18) ; Antisense N3, 5'-CGCGTCAGGA GCGCACCATC TTCTTCAAGT TAACTTGAAGAAGATGGTGC GCTCCTGCGA TCGGATCCTC TTAC-3' (SEQ.ID.NO.19); Sense N4, 5'-CGCGTTACTA ACTGGTACCTCTTCTTTTTT TTTTTGATAT CCTGCAG-3' (SEQ.ID.NO.20) ; Antisense N4, 5'-GTCCTGCAGG ATTCAAAAAAAAAAAGAAGAGGTACCAGTTAGTAA-3' (SEQ.ID.NO.21). All sequences listed from SEQ.ID.NO.14-SEQIDNO.21...

Embodiment 3

[0161] mir-302s cell culture and transgene delivery

[0162] Human cancer PC3 and Colo 829 cell lines were obtained from the American Culture Center (ATCC, Rockville, MD), while hHFC and hpESC cells were decomposed with collagenase / trypsin (4:1) to decompose 2 to 10 hair follicle roots or 2mm from hair or arm of the inventor 3 Prepared by explantation of the skin. at 37°C and 5% CO 2 These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 4mM L-glutamine, 1mM sodium pyruvate and 100μg / ml gentamicin (Sigma Chemical, MO). Cultures were passaged at 70-80% confluency by exposing cells to trypsin-EDTA solution for 1 min and rinsing once in RPMI, and then re-plating these detached cells at a 1:10 dilution into fresh growth medium. For transgenic mir-302s delivery by electroporation, the pTet-On-tTS-mir302s vector (10-30 μg) was mixed with the host cells (200-2000 μg) in a hypotonic pH buffer (400 μl; microcentrifuge tube) Mixing, and ele...

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Abstract

The present invention generally relates to a method for developing, generating and selecting tumor-free embryonic stem (ES)-like pluripotent cells using electroporation delivery of an inducible tumor suppressor mir-302 agent into mammalian cells. More particularly, the present invention relates to a method and composition for generating a Tet-On / Off recombinant transgene capable of expressing a manually re-designed mir-302 microRNA (miRNA) / shRNA agent under the control of doxycyclin (Dox) in human somatic / cancer cells and thus inducing certain specific gene silencing effects on the differentiation-associated genes and oncogenes of the cells, resulting in reprogramming the cells into an ES-like pluripotent state.

Description

[0001] Inventors: Lin Xilong and Wu Tangxi [0002] claim priority [0003] This application claims priority to US Provisional Application Serial No. 61 / 193,438, entitled "Use of Inducible Recombinant Ribonucleic Acids to Generate Tumor-Free Embryonic Stem Cell-Like Pluripotent Cells," filed November 28, 2008. This application claims priority to US Provisional Application Serial No. 61 / 011,333, entitled "Using Intronic RNA to Generate Pluripotent Human Embryonic Stem Cell-Like Cells," filed January 16, 2008. This application also claims priority to US Provisional Application Serial No. 61 / 191,327, entitled "Generation of Tumor-Free Embryonic Stem Cell-Like Pluripotent Cells Using Inducible Recombinant RNA Factors," filed September 8, 2008. Additionally, this application is a continuation-in-part of U.S. Application Serial No. 10 / 439,262, filed May 15, 2003, entitled "Directed RNA Splicing and Processing Gene Silencing and Related Applications"; March 2006 No. 11 / 278,143, entit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N5/06C12N5/074C12N15/113
CPCC12N2501/65C12N2501/40C12N5/0696C12N2510/00C12N2310/141C12N15/113
Inventor 林希龙吴堂熙
Owner 林希龙
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