Method of identifying genes controlling differentiation

• US 20030232358A1
• Filed: 03/14/2003
• Published: 12/18/2003
• Est. Priority Date: 03/15/2002
• Status: Abandoned Application

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A.
• Application Number:
  US10/389,120
• Publication Number:
  US 20030232358A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

A method for identifying the genetic factors responsible for cell differentiation based on expression cloning is described. To determine what genetic factor leads to the differentiation of a cell from a beginning cell type to a target cell type, a cDNA library is obtained, packaged in an expression vector and transformed into cells of the beginning cell type. The expression vector also preferable includes a marker gene system under the control of a tissue specific promoter operable in the tissue type of the target cell type. The transformed cells are then cultured until cell differentiation begins, then the culture is screened or selected to identify the cells which undergo differentiation toward the target cell type. By examining the cDNA insert in the differentiated cells, the genetic factor responsible for the differentiation process can be identified.

Claims

1. A method for identifying a genetic factor responsible for differentiation of a beginning cell to a target cell, the method comprising the steps of

obtaining a cDNA library representing genes expressed in the target cell type;

placing copies of the cDNA library into expression vectors which will express in the beginning cell type;

transforming the expression vectors into cells of the beginning cell type;

culturing cells of the beginning cell type until at least some cells differentiate;

identifying in the cultured cells at least one cell which has differentiated into the target cell type; and

identifying the cDNA in the cell which has differentiated to identify the genetic factor responsible for the cell differentiation

2. A method as claimed in claim 1 wherein the beginning cell type is a human undifferentiated stem cell.

3. A method as claimed in claim 1 wherein the expression vector is selected from the group consisting of an EBV vector and a lentivirus vector.

4. A method as claimed in claim 1 wherein the beginning cell also includes a marker system under the control of a tissue specific promoter, which causes tissue specific expression of the marker system in the target cell type, and further wherein the step of identifying the at least one cell is performed by identifying a cell expressing the marker system.

5. A method as claimed in claim 4 wherein the marker system is a selectable marker.

6. A method as claimed in claim 1 wherein the step of identifying the cDNA is performed by performing a PCR process on DNA recovered from the differentiated cell.

7. A method for identifying a genetic factor responsible for differentiation of a beginning cell to a target cell, the method comprising the steps of

making a cDNA library from mRNA from cells in the lineage of the target cell type;

placing copies of the cDNA library into expression vectors which will express in the beginning cell type;

transforming the expression vectors into cells of the beginning cell type;

culturing cells of the beginning cell type until at least some cells differentiate;

identifying in the cultured cells at least one cell which has differentiated into the target cell type; and

identifying the cDNA in the cell which has differentiated to identify the genetic factor responsible for the cell differentiation.

8. A method as claimed in claim 7 wherein the beginning cell type is a human undifferentiated stem cell.

9. A method as claimed in claim 7 wherein the expression vector is selected from the group consisting of an EBV vector and a lentivirus vector.

10. A method as claimed in claim 7 wherein the beginning cell also includes a marker system under the control of a tissue specific promoter, which causes tissue specific expression of the marker system in the target cell type, and further wherein the step of identifying the at least one cell is performed by identifying a cell expressing the marker system.

11. A method as claimed in claim 10 wherein the marker system is a selectable marker.

12. A method as claimed in claim 7 wherein the step of identifying the cDNA is performed by performing a PCR process on DNA recovered from the differentiated cell.