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Recombinant adenovirus vector for efficiently inducing pluripotent stem cell (PS cell), method for inducing PS cell by using recombinant adenovirus vector and usage of recombinant adenovirus vector

A technology of pluripotent stem cells and recombinant adenovirus, which is applied in the field of genetic engineering and stem cell science, can solve the problems of embryonic stem cell differentiation and the inability of embryos to form epiblasts

Active Publication Date: 2013-12-11
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the expression of Sox2 is not limited to pluripotent cells, it is also very important for early embryonic development and inhibition of differentiation. Embryos lacking Sox2 cannot form the epiblast, and blocking the function of Sox2 in embryonic stem cells results in the development of embryonic stem cells. differentiation

Method used

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  • Recombinant adenovirus vector for efficiently inducing pluripotent stem cell (PS cell), method for inducing PS cell by using recombinant adenovirus vector and usage of recombinant adenovirus vector
  • Recombinant adenovirus vector for efficiently inducing pluripotent stem cell (PS cell), method for inducing PS cell by using recombinant adenovirus vector and usage of recombinant adenovirus vector
  • Recombinant adenovirus vector for efficiently inducing pluripotent stem cell (PS cell), method for inducing PS cell by using recombinant adenovirus vector and usage of recombinant adenovirus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Taking the type 5 adenovirus vector as an example, the construction of the adenovirus right-arm plasmid pPE3-F11 containing the head coding sequence of the Ad11 adenovirus cilium is illustrated

[0090] Step 1: Synthesize (TAKARA) adenovirus type 11 ciliary protein gene coding sequence (sequence source: NC_011212, GenBANK) as shown in SEQ ID NO: 3, named Ad5F11, wherein: (1) 232-365 bases : type 5 adenovirus ciliary protein tail sequence; (2) bases 364-1212: type 11 adenoviral ciliary protein shaft and knob sequences; (3) the rest of the bases: type 5 adenovirus genome sequence, see Microbix Biosystems pBHGloxdeltaE1, 3Cre sequence.

[0091] Step 2: Synthesize the VT176 sequence (SEQ ID NO: 4) and the VT177 sequence (SEQ ID NO: 5) to construct the pCLON9 vector. The Ad5F11 sequence was digested with PacI+HindIII, and a 2656bp fragment was recovered, which was ligated with the vector digested with pCLON9 / PacI+HindIII, confirmed by enzyme digestion and sequenc...

Embodiment 2

[0095] Example 2: Construction of pDC328-OSN and pDC328-OKS

[0096] Step 1: Synthesize the EF1α promoter sequence (TAKARA), see SEQ ID NO: 7 for the sequence.

[0097] Step 2: The synthesized EF1 was double digested with XbaI+SalI, and a 600bp fragment was recovered.

[0098] Step 3: The pDC315 vector (purchased from Microbix Biosystem Inc. (Toronto), containing the 1417 to 2344bp sequence fragment of the left arm E1 region of type 5 adenovirus, and the inverted terminal repeat sequence) was digested with XbaI+SalI double enzymes, and the 3345bp fragment was recovered .

[0099] Step 4: Ligate the fragments recovered in Step 3 and Step 4, and confirm by enzyme digestion and sequencing (Invitrogen). The correct one named the vector as pDC328.

[0100] Step 5: Synthesize OCT4-2A-SOX2-2A-NANOG sequence (TAKARA), sequence source: OCT4 NM_002701; SOX2 NM_033106; NANOG NM_024865, GenBank. The sequence is shown in SEQ ID NO: 8, wherein: (1) bases 14-1093 are the coding sequence o...

Embodiment 3

[0105] Example 3: Construction of pDC328-EGFP and pDC315-EGFP

[0106] Step 1: Synthesize EGFP sequence (TAKARA), see SEQ ID NO:10 for the sequence.

[0107] Step 2: The synthesized EGFP was digested with EcoRI+SalI to recover a 726bp fragment.

[0108] Step 3: pDC328 and pDC315 were digested with EcoRI+SalI to recover 3897bp and 3883bp fragments respectively.

[0109] Step 4: The fragments recovered in the second step were ligated with the fragments recovered by digestion with pDC328 and pDC315 respectively.

[0110] The construction process of pDC328-EGFP and pDC315-EGFP is as follows Figure 5 shown.

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Abstract

The invention relates to a recombinant adenovirus vector for efficiently inducing a pluripotent stem cell (PS cell) and method for inducing the PS cell by using the recombinant adenovirus vector. The recombinant adenovirus vector is characterized in that the fiber genes therein are B subgroup adenovirus fiber genes, and an Sox2 gene expression cassette and an Oct4 gene expression cassette are connected within the recombinant adenovirus vector in an operating way. The terminally differentiated cell or the adult stem cell of the mammal, particularly the human is infected in vitro, the Oct4 genes and the Sox2 genes are expressed in an ectopic way, and the terminally differentiated cell or the adult stem cell of the mammal, particularly the human can be induced into the PS cell efficiently and rapidly under the synergistic effect of adjusting the epigenetic-inheritance small-molecular medicament. The PS cell of the human can be used for the cell replacement therapy to treat diseases, and the PS cell of the mammal can be used for preparing the transgenic animal model and the animal disease model.

Description

field of invention [0001] The invention relates to technologies in the fields of genetic engineering and stem cell science. In particular, it relates to a recombinant adenovirus with a modified adenovirus vector and an objective gene expression cassette, which can induce mammalian cells to become pluripotent stem cells (induced pluripotent stem cells). Background technique [0002] embryonic stem cells [0003] Embryonic stem cells (embryonic stem cells) come from the inner cell mass of embryos that have developed to the blastocyst stage. They are a group of totipotent cells with self-renewing ability. Under certain conditions, they can differentiate into various functional cells. The uses of embryonic stem cells are very extensive, involving many fields of medicine. The most far-reaching potential use of human embryonic stem cells may be the production of cells and tissues that can be stimulated to develop into specialized cells for the treatment of many diseases through ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N5/10A61K35/76A61K8/99A61P43/00A01K67/027C12R1/93A61K35/761
Inventor 钱其军周向军张琪彭新荣李琳芳吴孟超
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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