CreloxP technology, what is it

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Cre-loxP technology, what is it?
Cre recombinase protein from bacteriophage is a
Type 1 DNA topoisomerase. This is a protein that
cuts one strand of DNA and then re-anneals the
DNA. Cre binds DNA at a specific recognition
sequence not found in mammalian DNA. It is a 34
bp inverted repeat called a loxP site
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Cre-loxP technology, what is it?
When there are two loxP sites present (in the
same orientation), Cre cuts the DNA and
re-anneals it deleting the intervening sequence.
loxP
loxP
Your gene of interest
Critical step, controlling Cre expression!
Cre
loxP
loxP
degraded
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Tricks to monitor Cre expression ROSA26
ROSA reverse orientation splice
acceptor Transfected this into ES cells, made
mice..
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ROSA alleles
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What IS ROSA26?
Thumpd3, antisense transcript from the ROSA26
locus. Predicted RNA methyltransferase activity.
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Tricks to monitor Cre expression ROSA26R
Soriano, P. 1999. Nature Genetics 21, 70-71.
STOP
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Tricks to monitor Cre expression ROSA26R
Soriano, P. 1999. Nature Genetics 21, 70-71.
Cre turns on lacZ in the ROSA26 locus. This
allele is often called R26R, or (incorrectly)
Rosa26.
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Tricks to monitor Cre expression ROSA26R
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Tricks to monitor Cre expression ROSA26R YFP or
CFP
Srinivas et al., 2001.
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Specialized versions of Cre Cre-ERT2 or Cre-ERTM
Tamoxifen (injected into pregnant mother or added
to cell culture medium)
Cre-ERT2
cytoplasm
nucleus
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Today, for observation Wnt1-Cre R26R embryos
9.5-13.5 dpc
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And then a quiz of sorts.
How old am I???
Credit MRC Human Genetics Unit.
Credit Gloria Kwon, nikonsmallworld.com
Credit jax.org