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=='''Cre-Lox Technology'''==
 
=='''Cre-Lox Technology'''==
Cre-Lox Technology provides a sophisticated means to selectively express a given gene by creating knockouts, conditional knockouts, and reporter strains in a variety of organisms from plants to mice.
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Cre-Lox Technology provides a sophisticated means to selectively express a given gene by creating knockouts, conditional knockouts, and reporter strains in a variety of organisms from plants to mice.
 
 
 
 
 
==='''How does this technology work?'''===
 
==='''How does this technology work?'''===
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<u>'''Putting the system together:'''[[File:Cre-lox.jpg|thumb|500px|The Cre Lox System in Mice. From the Science Creative Quarterly, Article: TARGETING YOUR DNA WITH THE CRE/LOX SYSTEM By Alfred Pechisker (Aug 2004)
 
<u>'''Putting the system together:'''[[File:Cre-lox.jpg|thumb|500px|The Cre Lox System in Mice. From the Science Creative Quarterly, Article: TARGETING YOUR DNA WITH THE CRE/LOX SYSTEM By Alfred Pechisker (Aug 2004)
 
http://www.scq.ubc.ca/targeting-your-dna-with-the-crelox-system/]]</u>
 
http://www.scq.ubc.ca/targeting-your-dna-with-the-crelox-system/]]</u>
Researchers will select an organism and a target gene they wish to study. Two lox sequences will then be inserted at both ends of the target gene. This DNA will be incoporated into one mouse. The researchers will then decide the conditions under which they want their target gene expressed (all the time vs. part of the time) and based the conditions they will pick another gene and incorporate the Cre gene downstream. This DNA will be incorporated into a second mouse. The two mice will breed and the offspring will have both the cre and loxP sequences. Whenever the promoter preceding the Cre sequence is transcribed, Cre will also be transcribed and later expressed. The expressed Cre will then cleave inbetween both loxP sequences encompassing the target gene and remove the target gene sequence [[File:Loxp_sites.jpg|thumb|500px|Mutations due to varied orientation of the loxp sites. Cre/loxP-Mediated Chromosome Engineering
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Researchers will select an organism and a target gene they wish to study. Two lox sequences will then be inserted at both ends of the target gene. This DNA will be incoporated into one mouse. The researchers will then decide the conditions under which they want their target gene expressed (all the time vs. part of the time) and based the conditions they will pick another gene and incorporate the Cre gene downstream. This DNA will be incorporated into a second mouse. The two mice will breed and the offspring will have both the cre and loxP sequences. Whenever the promoter preceding the Cre sequence is transcribed, Cre will also be transcribed and later expressed. The expressed Cre will then cleave inbetween both loxP sequences encompassing the target gene and remove the target gene sequence creating a knock-out mouse.
of the Mouse Genome.V. Brault · V. Besson · L. Magnol · A.Duchon · Y. Hérault]]creating a knock-out mouse.
 
   
  +
&nbsp;
'''Generation of DIfferent Mutations.&nbsp; '''Depending on the orientation of the loxp sites different mutations can be generated.&nbsp; As you can see to the left Deletions, Duplications, and Inversions are some of the mutations researchers can generate.
 
  +
  +
'''Generation of DIfferent Mutations.&nbsp; [[File:Loxp_sites.jpg|thumb|500px|Mutations due to varied orientation of the loxp sites. Cre/loxP-Mediated Chromosome Engineering
 
of the Mouse Genome.V. Brault · V. Besson · L. Magnol · A.Duchon · Y. Hérault]]'''
  +
 
Depending on the orientation of the loxp sites different mutations can be generated.&nbsp; As you can see to the left Deletions, Duplications, and Inversions are some of the mutations researchers can generate.
  +
  +
&nbsp;
   
 
'''<u>History</u>'''
 
'''<u>History</u>'''
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<u>'''Overview of P1 and the Cre Lox System '''</u>
 
<u>'''Overview of P1 and the Cre Lox System '''</u>
   
The bacteriophage DNA is double stranded with a molecular weight of 6.6 x 107, unlike other phages P1 has a linear genetic map. Within the paper, the authors wanted to evaluate the recABC independent recombination on the EcoRI fragment 7. In doing so , they uncovered the Cre enzyme and the loxP regions.
+
The bacteriophage DNA is double stranded with a molecular weight of 6.6 x 107, unlike other phages P1 has a linear genetic map. Within the paper, the authors wanted to evaluate the recABC independent recombination on the EcoRI fragment 7. In doing so , they uncovered the Cre enzyme and the loxP regions.
 
<u>'''Overview of Cre-lox first used in Yeast'''</u>
 
<u>'''Overview of Cre-lox first used in Yeast'''</u>
   
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'''<u>Overview of the Cre-lox system first used in Mammalian Cells</u>'''
 
'''<u>Overview of the Cre-lox system first used in Mammalian Cells</u>'''
   
Researchers, Dr. Brian Sauer and Dr. Nancy Henderson, successfully applied the Cre-Lox system in mouse cell line (C-127) . The cre gene was downstream a MT-I (or mouse metallothionein gene) and upstream from BPV(bovine papilloma virus) a sequence that would enable extrachromosomal replication of the plasmid in the mouse cell. The loxP sites encompassed kan r gene in a plasmid pRH43 (Kan r stands for Kanamycin resistant).
+
Researchers, Dr. Brian Sauer and Dr. Nancy Henderson, successfully applied the Cre-Lox system in mouse cell line (C-127) . The cre gene was downstream a MT-I (or mouse metallothionein gene) and upstream from BPV(bovine papilloma virus) a sequence that would enable extrachromosomal replication of the plasmid in the mouse cell. The loxP sites encompassed kan r gene in a plasmid pRH43 (Kan r stands for Kanamycin resistant).
 
&nbsp;
 
&nbsp;
   
 
<u>Fields that have used the Cre-lox System</u>
 
<u>Fields that have used the Cre-lox System</u>
   
-This use of this system in mice is widespread enabling scientists to study everything from the inner ear to cancers.
+
-This use of this system in mice is widespread enabling scientists to study everything from the inner ear to cancers.
 
&nbsp;
 
&nbsp;
   

Revision as of 21:10, 31 October 2012

Cre-Lox Technology

Cre-Lox Technology provides a sophisticated means to selectively express a given gene by creating knockouts, conditional knockouts, and reporter strains in a variety of organisms from plants to mice.  

How does this technology work?

This technology is contingent upon the presence of two genes Cre and loxP.

Cre Protein: The Cre protein is also known as cyclization recombination protein. It is a 38 kDa protein natively found in the bacterophage P1 as the gene cre. The target substrate for Cre are the loxP regions. Cre will identify pairs of loxP sites and then catalyze reciprocal DNA recombination between the two sites, often resulting in the excision of a small piece of DNA. Cre will only catalyze the recombinantion of two consecutively expressed loxP sites. (In other words, there will not be a loxP site intervening in the middle of the sequence.)

In bacteriophage P1 native Cre has 2 important roles (1) after infection it acts as a backup mechanism for cycling the DNA. (2) Increases the stability of P1 by facilitating separation between dimeric plasmids during bacterial division.

Important to note, Cre is NOT the same as CRE (cAMP response element) found in eukaryotes.

LoxP:A loxP site is typically 34bp site. This 34 bp site contains two 13bp inverted repeats with an 8 bp core. The core is a non-pallindromic sequence. All of the 34 bp are not necessary for successful recombination by Cre, some of the base pairs can be modified.

Putting the system together:

Cre-lox

The Cre Lox System in Mice. From the Science Creative Quarterly, Article: TARGETING YOUR DNA WITH THE CRE/LOX SYSTEM By Alfred Pechisker (Aug 2004) http://www.scq.ubc.ca/targeting-your-dna-with-the-crelox-system/

Researchers will select an organism and a target gene they wish to study. Two lox sequences will then be inserted at both ends of the target gene. This DNA will be incoporated into one mouse. The researchers will then decide the conditions under which they want their target gene expressed (all the time vs. part of the time) and based the conditions they will pick another gene and incorporate the Cre gene downstream. This DNA will be incorporated into a second mouse. The two mice will breed and the offspring will have both the cre and loxP sequences. Whenever the promoter preceding the Cre sequence is transcribed, Cre will also be transcribed and later expressed. The expressed Cre will then cleave inbetween both loxP sequences encompassing the target gene and remove the target gene sequence creating a knock-out mouse.

 

Generation of DIfferent Mutations. 

Loxp sites

Mutations due to varied orientation of the loxp sites. Cre/loxP-Mediated Chromosome Engineering of the Mouse Genome.V. Brault · V. Besson · L. Magnol · A.Duchon · Y. Hérault

Depending on the orientation of the loxp sites different mutations can be generated.  As you can see to the left Deletions, Duplications, and Inversions are some of the mutations researchers can generate.

 

History The Cre-lox genes were first discovered in P1 bacteriophage in the 1980's. It was discovered that these genes were a part of the virus' normal life cycle and were used to facilitate DNA replication and enable the genomic DNA to circularize. Since then site-specific DNA recombinases have also been observed including in yeast.

Historic Papers of Note -Bacteriophage Pl Site-specific Recombination I. Recombination between loxP Sites. Nat Sternberg, Daniel Hamilton. (1981) - Discovery of the Cre-lox system in bacteriophage P1.

- Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.- Sauer B. (1987) - first use of Cre recombinase in yeast.

- Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1- Sauer B, Henderson N (1988) - first use of Cre recombinase in mammalian cells.

-Tissue-and site-specific DNA recombination in transgenic mice - Paul Orban, Daniel Chui, Jamey Marth (1992) - first use of Cre-loxP mouse.

Overview of P1 and the Cre Lox System

The bacteriophage DNA is double stranded with a molecular weight of 6.6 x 107, unlike other phages P1 has a linear genetic map. Within the paper, the authors wanted to evaluate the recABC independent recombination on the EcoRI fragment 7. In doing so , they uncovered the Cre enzyme and the loxP regions. Overview of Cre-lox first used in Yeast

Researcher Brian Sauer had noted in some instances that bacterial DNA enzymes retained the ability to act on eukaryotic DNA, despite a variety of significant differences between the two types of cells. Having acknowledged this, Dr. Sauer decided to investigate whether a bacteria recombination system could be used in the eukaryotic cell. He selected the cre-lox site specific recombination system found in coliphage P1 (bacteriophage that infects E.coli). This paper was the first paper where researchers began to see the potential to use the cre-lox system in other eukaryotes for site specific recombination.

Crelox yeast2

Generating plasmids expressing the Cre. (Functional Expression of the cre-lox Site-Specific Recombination System in the Yeast Saccharomyces cerevisiae. Brian Sauer (1987), Figure 1)

What they actually did in the paper:

Figure to the left demonstrates the construction of the cre gene downstream the yeast galactose gene GALI. To accomplish this they obtained pBS7, which contains the cre gene. From this they were able perform restriction digests to pBS7 to exise the cre gene. They also digested the pBM150 (right side of the figue), which contained the native GALI gene and were able to ligate the two together. They also incorporated cre upstream of a mouse metallothionein gene (the left side of the figure) . The resulting plasmid, pBS49, contained the GALI gene upstream cre followed by the mouse metallothionein gene.

Restriction Digests and ligations were also used to incorporate the loxP sites around the LEU2 gene.

Overview of the Cre-lox system first used in Mammalian Cells

Researchers, Dr. Brian Sauer and Dr. Nancy Henderson, successfully applied the Cre-Lox system in mouse cell line (C-127) . The cre gene was downstream a MT-I (or mouse metallothionein gene) and upstream from BPV(bovine papilloma virus) a sequence that would enable extrachromosomal replication of the plasmid in the mouse cell. The loxP sites encompassed kan r gene in a plasmid pRH43 (Kan r stands for Kanamycin resistant).  

Fields that have used the Cre-lox System

-This use of this system in mice is widespread enabling scientists to study everything from the inner ear to cancers.  

Very Start of Works Cited(more to be added):

Review Article: Inducible Gene Targeting in Mice Using the Cre/lox System. Brian Sauer (1998)

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.- Sauer B. (1987) - first use of Cre recombinase in yeast.

Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1- Sauer B, Henderson N (1988)

Bacteriophage Pl Site-specific Recombination I. Recombination between loxP Sites. Nat Sternberg, Daniel Hamilton. (1981) - Discovery of the Cre-lox system in bacteriophage P1.