US 2008/0293143 A1 · Generation of human embryonc stem-like cells using intronic RNA

Abstract

This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.

Claims61

Dependent claims

A transgenic method for inducing intronic mir-302-mediated gene silencing effects in mammalian cells comprises the steps of:

(a) Constructing a recombinant nucleic acid composition that contains at least an intron encoding a mir-302-like gene-silencing effector flanked with exons, wherein said intron can be cleaved out of the exons for inducing mir-302-mediated gene silencing;
(b) Cloning said recombinant nucleic acid composition into an expression-competent vector;
and
(c) Introducing said expression-competent vector into a plurality of mammalian cells, wherein said cells generate a plurality of primary RNA transcripts of said recombinant nucleic acid composition, and wherein the cells splice said intron out of the primary RNA transcripts so as to provide a mir-302-mediated gene silencing effect on genes containing complementarity to the sequence of the mir-302-like gene-silencing effector, which consequently results in reprogramming the cells into a plurality of stem cell-like pluripotent cells.

1 more element

Dependencies are inferred from the claim text.

Generation of human embryonc stem-like cells using intronic RNA

Life of this patent

Generation of human embryonc stem-like cells using intronic RNA

Life of this patent

Abstract

Claims61