Nucleic acids encoding FOXD3 promoter and methods to isolate FOXD3 expressing cells
DNA enhancer sequences are provided for use in constructs to identify early stage embryonic cells. The enhancer sequences can be used in parallel with short-hairpin RNA in a vector construct for endogenously regulated gene knockdowns. The disclosed enhancer sequences can be used to isolate a selected population of early stage embryonic cells.
1. An isolated nucleic acid sequence consisting of SEQ ID NO: 4.
2. A nucleic acid vector for down-regulating gene expression comprising:
a short-hairpin RNA sequence under transcriptional control of at least one enhancer sequence having the sequence as set forth in SEQ ID NO: 4.
3. An expression vector comprising the sequence of SEQ ID NO: 4 and a nucleic acid sequence encoding a reporter protein.
4. The expression vector of claim 3 , wherein the reporter protein is a fluorescent protein.
5. The expression vector of claim 3 , wherein the expression vector is a viral vector.
6. A method of isolating mammalian cells expressing FoxD3, the method comprising:
introducing to a population of isolated mammalian cells an expression vector comprising the sequence of SEQ ID NO: 4 and a nucleic acid sequence encoding a reporter protein; and
identifying introduced cells that produce the reporter protein.
7. The method of claim 6 , wherein the reporter protein is a fluorescent protein.
8. The method of claim 6 , further comprising isolating the cells expressing the reporter protein.
9. The method of claim 6 , wherein the isolating is performed by flow cytometry.