• loxP sites: These are 34-base-pair DNA recognition sequences. A loxP-containing ("floxed") gene or DNA segment is flanked by these sites. The orientation of the loxP sites determines the outcome of recombination.

• Cre recombinase: This enzyme, derived from bacteriophage P1, recognizes the loxP sites and catalyzes DNA recombination.

• Conditional gene knockout: This is one of the most common applications.

  • A cell line is created with loxP sites flanking a gene of interest. The gene functions normally until Cre is introduced.

  • The introduction of Cre recombinase causes the gene segment between the two loxP sites to be excised, thereby knocking out the gene in those specific cells.

  
  

• Inducible activation or knockout: For greater control, the Cre recombinase is linked to a modified estrogen receptor (CreER).

  • CreER is inactive and sequestered in the cytoplasm until a small molecule, like tamoxifen (TAM), is added.

  • Upon activation by tamoxifen, the CreER fusion protein moves to the nucleus and performs recombination. This allows researchers to control the exact timing of gene modification.

  
  

• Lineage tracing: This technique uses a lox-stop-lox cassette placed before a reporter gene, such as a fluorescent protein (e.g., GFP or tdTomato).

  • In the absence of Cre, the "stop" sequence prevents the reporter from being expressed.

  • In the presence of Cre, the stop sequence is excised, and the reporter gene is expressed. This permanently labels the cell and all of its descendants with the fluorescent protein, allowing researchers to track the fate of specific cell populations.

  
  

• Cell-specific gene targeting: To restrict genetic changes to a particular cell type, the Cre recombinase gene can be placed under the control of a tissue-specific promoter.

  • This ensures that Cre is only expressed in a specific type of cell, such as neurons or cardiac muscle cells.

  • When Cre-expressing cells are bred with cells containing floxed genes, the recombination only occurs in the target cell population, leaving other cells unaffected.

  
  

• H9 Cre-LoxP cell line: WiCell, a research institute, offers a modified H9 human embryonic stem cell (hESC) line that has been engineered with a Cre-Lox site. This line also constitutively expresses GFP, allowing for tracing after further gene editing.

• Plasmids and viral systems: Researchers also create custom lox-Cre-loxP systems using plasmids or viral delivery systems to introduce Cre recombinase into various mammalian cell lines, including human cells.

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