Embryonic stem cell self maintenance and renewal reporter
View Patent ↗The present invention relates to methods and compositions for assaying embryonic stem cell maintenance. In particular, the present invention provides reporter constructs for stem cell pluripotency and differentiation and cells and organisms containing such constructs.
1. A composition comprising a bacterial artificial chromosome (BAC), wherein said BAC comprises
a) a reporter gene having a 5′ end and a 3′ end,
b) a first Nanog gene regulatory sequence operably linked to said 5′ end of said reporter gene, wherein said first Nanog gene regulatory sequence is produced by amplification of a mouse DNA sequence using a first PCR forward primer (SEQ ID NO:3) and a first PCR reverse primer (SEQ ID NO:4) in a first polymerase chain reaction, and
c) a second Nanog gene regulatory sequence operably linked to said 3′ end of said reporter gene, wherein said second Nanog gene regulatory sequence is produced by amplification of a mouse DNA sequence using a second PCR forward primer (SEQ ID NO:5) and a second PCR reverse primer (SEQ ID NO:6) in a second polymerase chain reaction,
wherein the level of expression of said reporter gene is lower in a differentiated mouse embryonic stem cell comprising said BAC compared to the level of expression of said reporter gene in an undifferentiated mouse embryonic stem cell comprising said BAC.
2. The composition of claim 1 , wherein said reporter gene encodes a fluorescent protein.
3. The composition of claim 2 , wherein said fluorescent protein is selected from the group consisting of green and red fluorescent protein.
4. The composition of claim 1 , wherein said BAC further comprises a selectable marker.
5. An isolated embryonic stem cell comprising the composition of claim 1 .
6. The isolated embryonic stem cell of claim 5 , wherein said isolated embryonic stem cell is selected from the group consisting of an isolated mouse embryonic stem cell and an isolated human embryonic stem cell.
7. An isolated embryonic germ cell comprising the composition of claim 1 .
8. An isolated primordial germ cell comprising the composition of claim 1 .
9. A composition comprising a portion of a Nanog gene, said portion is produced by amplification of a mouse DNA sequence using a first PCR forward primer (SEQ ID NO:3) and first PCR reverse primer (SEQ ID NO:4) in a polymerase chain reaction.
10. A composition comprising a portion of a Nanog gene, said portion is produced by amplification of a mouse DNA sequence using a first PCR forward primer (SEQ ID NO:5) and first PCR reverse primer (SEQ ID NO:6).
11. The composition of claim 1 , wherein said first Nanog gene regulatory sequence comprises a nucleotide sequence from nucleotide 9 to nucleotide 539 of SEQ ID NO:55.
12. The composition of claim 1 , wherein said second Nanog gene regulatory sequence comprises a nucleotide sequence from nucleotide 2523 to nucleotide 3089 of SEQ ID NO:55.
13. A composition comprising a bacterial artificial chromosome (BAC), wherein said BAC comprises
a) a reporter gene having a 5′ end and a 3′ end,
b) a first Nanog gene regulatory sequence from nucleotide 9 to nucleotide 539 of SEQ ID NO:55, wherein said first Nanog gene regulatory sequence is operably linked to said 5′ end of said reporter gene, and
c) a second Nanog gene regulatory sequence from nucleotide 2523 to nucleotide 3089 of SEQ ID NO:55, wherein said second Nanog gene regulatory sequence is operably linked to said 3′ end of said reporter gene.
14. The composition of claim 13 , wherein said reporter gene encodes a fluorescent protein.
15. The composition of claim 13 , wherein said fluorescent protein is selected from the group consisting of green fluorescent protein and red fluorescent protein.
16. The composition of claim 13 , wherein said BAC further comprises a selectable marker.
17. An isolated embryonic stem cell comprising the composition of claim 13 .
18. The isolated embryonic stem cell of claim 17 , wherein said isolated embryonic stem cell is selected from the group consisting of an isolated mouse embryonic stem cell and an isolated human embryonic stem cell.
19. An isolated embryonic germ cell comprising the composition of claim 13 .
20. An isolated primordial germ cell comprising the composition of claim 13 .
21. A composition comprising a nucleic acid consisting essentially of nucleotide 9 to nucleotide 539 of SEQ ID NO:55.
22. A composition comprising a nucleic acid consisting essentially of nucleotide 2523 to nucleotide 3089 of SEQ ID NO:55.