ASSESSING EXPRESSION OF ENDOGENOUS AND EXOGENOUS GENES

• US 20100167291A1
• Filed: 08/11/2009
• Published: 07/01/2010
• Est. Priority Date: 09/12/2008
• Status: Abandoned Application

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Rosenberg, Martin, Storts, Douglas
• Application Number:
  US12/539,436
• Publication Number:
  US 20100167291A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The present invention relates to compositions and methods to assess gene expression in cells. In particular, the present invention provides compositions and methods to assess expression from an exogenous gene and an endogenous version of the same gene in induced pluripotent stem cells.

Claims

1. A method comprising:

a) providing;

i) one or more cells;

ii) an exogenous gene encoding a de-differentiation factor, said exogenous gene differing in sequence from an endogenous version of said gene in said one or more cells;

b) introducing said exogenous gene into said one or more cells to induce de-differentiation of said one or more cells; and

c) detecting the presence of, absence of, or amount of expression of said exogenous gene and said endogenous gene.

2. The method of claim 1, wherein said exogenous gene and said endogenous gene express protein of identical amino acid sequence.

3. The method of claim 1, wherein said discriminating is based on a detectable property in the mRNA of said exogenous gene and said endogenous gene.

4. The method of claim 3, wherein said detectable property is a difference in mRNA sequence of the exogenous gene and said endogenous version of the same gene.

5. The method of claim 4, wherein said difference in mRNA sequence occurs in the wobble position of one or more mRNA codons.

6. The method of claim 4, wherein said difference in mRNA sequence occurs in an untranslated region of said mRNA.

7. The method of claim 3, wherein said discriminating of said detectable property comprises reverse transcription of said mRNA of said exogenous gene and said endogenous gene.

8. The method of claim 3, wherein said discriminating of said detectable property comprises amplification of said exogenous gene and said endogenous gene.

9. The method of claim 3, wherein said discriminating of said detectable property comprises hybridization of one or more probes to said exogenous gene and said endogenous gene.

10. The method of claim 1, wherein said cells are induced pluripotent stem cells.

11. The method of claim 1, wherein said de-differentiation factor is selected from the group consisting of:

Oct1, Oct3, Oct4, Oct3/4, Oct6, Sox1, Sox2, Sox3, Sox15, Sox18, Nanog, C-Myc, N-Myc, L-Myc, Klf1, Klf2, Klf4, Klf5, LIN28, and Fbx15.

12. The method of claim 1, further comprising the step of treating the cells with an agent after step b), but prior to step c).

13. A composition comprising:

a) a synthetic, non-native de-differentiation gene having a sequence that differs from a native de-differentiation gene; and

b) a reagent that detects an expression product of said synthetic, non-native de-differentiation gene and differentiates said expression product from an expression of said native de-differentiation gene.

14. The composition of claim 13, wherein said de-differentiation gene is selected from the group consisting of:

Oct1, Oct3, Oct4, Oct3/4, Oct6, Sox1, Sox2, Sox3, Sox15, Sox18, Nanog, C-Myc, N-Myc, L-Myc, Klf1, Klf2, Klf4, Klf5, LIN28, and Fbx15.

15. The composition of claim 13, wherein said reagent comprises one or more nucleic acid probes that binds specifically to said expression product from said synthetic, non-native de-differentiation gene relative to said native gene.

16. The composition of claim 13, wherein said reagent comprises one or more primer probes that binds specifically to said expression product from said synthetic, non-native de-differentiation gene relative to said native gene.

17. The composition of claim 13, wherein synthetic gene is present in an expression vector.

18. The composition of claim 17, wherein said expression vector is a viral expression vector.