Difference between revisions of "Part:BBa K1926002"
(→‎Usage)
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===Usage===  
 
===Usage===  
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 +
You may use this part to:
  
 
1) Express something in mammal cell lines particularly in G1 phases or once in every cell cycle by stable transfecting it into cell line;
 
1) Express something in mammal cell lines particularly in G1 phases or once in every cell cycle by stable transfecting it into cell line;
  
 
2) Use it as a human promoter by transient transfecting it into cells.
 
2) Use it as a human promoter by transient transfecting it into cells.
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===Source===
 
===Source===

Revision as of 08:56, 11 October 2016

A cyclic promoter of Ki-67 from human genome

This part is a cyclic promoter of Ki67 from human genome. It can lead to transcription of the downstream DNA sequence once in every G1 phase[1].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 425
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 422


Usage

You may use this part to:

1) Express something in mammal cell lines particularly in G1 phases or once in every cell cycle by stable transfecting it into cell line;

2) Use it as a human promoter by transient transfecting it into cells.

Source

The sequence was retrieved from Addgene. We got it from human genome through PCR using the following primers:

pKi67-F: ACCTCTGCCCTCCGCCAGCCG

pKi67-R: ACCCGGTGGCCCTACAGGCTACG

(Product: 360)


Promoter function confirmation

G1 promoter function confirmation by transient transfection using 293T cells. Photos taken 48 hours after transient transfection, 10x. pCDK4, pKi67 and pCCNE are our G1 promoters. pmPGK is the constitutive promoter of mouse PGK, it is a medium promoter, here used as a control.

Figure 1: in vivo testing pG1 promoter function in human 293 cell line.



Reference

1. Pei, D.S., et al., Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription. Tumour Biol, 2012. 33(1): p. 257-66.