Gene transfer into primate embryonic stem cells using VSV-G pseudo type simian immunodeficiency virus vectors

A technology of immunodeficiency virus and embryonic stem cells, applied to embryonic cells, using vectors to introduce foreign genetic material, viruses/bacteriophages, etc., can solve problems such as difficulties in gene introduction

Inactive Publication Date: 2005-01-26
DNAVEC RES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no reports on the introduction of genes into primate ES cells, only reports at academic conferences point out that gene introduction into primate ES cells is more difficult than gene introduction into mouse ES cells

Method used

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  • Gene transfer into primate embryonic stem cells using VSV-G pseudo type simian immunodeficiency virus vectors
  • Gene transfer into primate embryonic stem cells using VSV-G pseudo type simian immunodeficiency virus vectors
  • Gene transfer into primate embryonic stem cells using VSV-G pseudo type simian immunodeficiency virus vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] [Example 1] Construction of SIV vector

[0101] The non-pathogenic African green monkey (African green monkey) immunodeficiency virus clone - SIVagmTYO1 was used to construct the vector system. figure 1 Represents an outline of the vector system. The sequence numbers of all nucleotides are represented by the transcription start point of viral RNA+1 below. As a plasmid, pSA212 inserted with SIVagmTYO1 was used (J. Viol., vol. 64, pp307-312, 1990). And all ligation reactions were carried out according to the instructions attached to Ligation High (Toyobo).

[0102] a. Construction of packaging vector

[0103]First, use primers 1F (SEQ ID NO: 1) and 1R (SEQ ID NO: 2) and use pSA212 as a template to obtain the region (5337-5770) corresponding to the first exon of tat / rev and vif by PCR of DNA fragments. An EcoRI restriction enzyme site was added to the PCR primer, thereby preparing a DNA fragment with an EcoRI site at the 3' end. After digestion with BglII and EcoR...

Embodiment 2

[0107] [Example 2] Modification of 5'LTR

[0108] The 5'LTR transcriptional activity of lentiviruses generally depends on the presence of Tat protein, a virus-derived factor. Therefore, in order to eliminate the dependence on Tat and increase the titer of the vector by substituting it with a highly transcriptionally active promoter sequence, a SIVagm gene transfer vector was prepared in which the 5'LTR promoter sequence--U3 region was replaced by other Promoter sequence replacement ( figure 2 ).

[0109] The 5'LTR was replaced with a chimeric promoter by PCR amplification using primers 9-1F-3F (SEQ ID NO: 21-23) and primer 9R (SEQ ID NO: 24) using pSA212 as a template Fragment comprising the region downstream of the TATA box of the 5'LTR up to the gag region (9039-9170+1-982). Then, a fragment containing the CMVL promoter (from pCI (Promega), 1-721) was amplified by PCR using primers 10-1F (SEQ ID NO: 25) and 10-1R (SEQ ID NO: 26) using pCI as a template ; A fragment comp...

Embodiment 3

[0110] [Example 3] Modification of 3'LTR

[0111] A partial sequence of the 3'LTR is excised to prevent full-length vector mRNA from being transcribed in target cells, thereby constructing a self-inactivating (SIV) vector with improved safety. In the lentiviral vector, it has been proved that the promoter sequence contained in the 3'LTR region - the U3 region can be integrated into the U3 promoter region of the 5'LTR during reverse transcription in the target cell. Therefore, in the genome of the target cell, the gene The U3 region contained in the 3'LTR region of the transfer vector plasmid can be used as the 5'LTR U3 promoter region associated with gene expression ( image 3 ). Therefore, a vector in which the 3'LTR U3 region of the SIVagm gene transfer vector was substituted with other promoter sequences was prepared ( image 3 ). In addition, in order to study whether the 5'LTR in the target cells can delete the promoter sequence, a vector that deletes the 3'LTR U3 regi...

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Abstract

Highly efficient gene transfer into primate-derived embryonic stem (ES) cells has successfully been achieved by using a simian immunodeficiency virus vector (SIV) pseudotyped with VSV-G protein, which is a surface glycoprotein of vesicular stomatitis virus (VSV) The present invention provides simian immunodeficiency virus vectors for gene transfer to primate ES cells. The method for gene transfer to primate ES cells using the vectors of the present invention is useful in, for example, research into embryology and disease, clinical applications, and experimental models for primates. The method is also useful in assaying and screening for genes and reagents able to enhance the specific differentiation of tissues or cells, and which are useful in preparing desired cells or tissues differentiated from ES cells.

Description

technical field [0001] The present invention relates to a simian immunodeficiency virus vector for introducing genes into primate embryonic stem cells. Background technique [0002] Embryonic stem cells (hereinafter referred to as ES cells) are pluripotent undifferentiated cells capable of autonomous replication. ES cells are known to have tissue repair ability after injury, so ES cells can be used to screen therapeutic substances for various diseases and in the field of regenerative medicine, and are currently being extensively studied. Compared with mouse ES cells, monkey ES cells are closer to human ES cells, so they are expected to be applicable to human disease models. [0003] The genetic engineering of ES cells is extremely important for its future application in the treatment of various diseases and injuries. In order to change the characteristics of ES cells such as proliferation ability or differentiation ability, or drug sensitivity, it is often necessary to sta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/85C12N15/867G01N33/50
CPCA01K2227/105A01K2217/05C12N2810/6081C12N2510/00C12N2740/15043A01K2267/03G01N33/5008C12N2740/15045G01N33/5073C12N15/8509C12N2506/02A01K2227/106G01N2333/22A61K48/00C12N15/86A01K2267/025
Inventor 花园丰上田泰次近藤靖铃木丰
Owner DNAVEC RES
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