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Cassette exchange article

Definitions

cassetteexchange.jpg

 
from cancer web dictionary :
cassette: A pre existing structure into which an insert can be moved. Fashionably used to refer to certain vectors. See: cassette mechanism.
vectors:<molecular biology> Commonly term for a plasmid that can be used to transfer DNA sequences from one organism to another. Different vectors may have properties particularly appropriate to give protein expression in the recipient or for cloning or may have different selectable markers.
Recombinant DNA systems especially suited for production of large quantities of specific proteins in bacterial, yeast, insect, or mammalian cell systems.
Recombinant DNA : Spliced DNA formed from two or more different sources that have been cleaved by restriction enzymes and joined by ligases.
cassette mechanisme:<molecular biology> Term used for genes such as the a and _ genes that determine mating type in yeast, either one or the other is active. In this gene conversion process, a double stranded nuclease makes a cut at a specific point in the MAT locus, the old gene is replaced with a copy of a silent gene from one or other flanking region and the new copy becomes active. As the process involves replacing one ready made construct with another in an active slot it is called a cassette mechanism.

recombination

<molecular biology> The creation, by a process of intermolecular exchange, of chromosomes combining genetic information from different sources, typically two genomes of a given species. Site specific, homologous, transpositional and nonhomologous illegitimate) types of recombination are known. 1st ed

from drug discovery & developement glossary :
Cassette Genetic material that is spliced into a genome.

alleles :Definition:Alternative forms of a genetic locus; a single allele for each locus is inherited separately from each parent (e.g., at a locus for eye color the allele might result in blue or brown eyes). Science and Biotechnology site

A gene is the basic physical and functional unit of heredity. Genes, which are made up of DNA, act as instructions to make molecules called proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases. The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes.

Every person has two copies of each gene, one inherited from each parent. Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people. Alleles are forms of the same gene with small differences in their sequence of DNA bases. These small differences contribute to each person’s unique physical features. Table of Contents genetic home reference http://ghr.nlm.nih.gov/info=basics/show/gene

recombinant DNA
A variety of techniques that molecular biologists use to manipulate DNA molecules to study the expression of a gene. (take dna from an organism, manipulate it, insert it in another organism.) geonome.gov

Homologous recombination

http://www.cellectis.com/homologous.html

In nature, homologous recombination is a DNA maintenance pathway that protects chromosomes against damage affecting both DNA strands, such as double strand breaks (DSBs) or interstrand crosslinks. DSB repair (DSBR) has been one of the most investigated homologous repair pathways (see DSBR web page).

The recombination machinery has been well conserved throughout evolution, as an essential component of cell survival. In addition to its maintenance role, homologous recombination underlies many biological pathways. It is involved in meiotic crossovers, which are responsible for the rearrangement of alleles, as well as being necessary for proper chromosome segregation. It is important for mating type switching in yeast and epitope class switching in many organisms. Finally, it is involved in the spreading of many mobile genetic elements such as P elements in Drosophila, and group I introns and inteins.

Interestingly, group I introns and inteins code for sequence-specific endonucleases, called homing endonucleases, which trigger homologous recombination events by delivering DSBs to their target sequences (see the meganuclease web page).

Homologous recombination has significantly contributed to genome engineering, because it is the basis of homologous gene targeting. Homologous gene targeting remains the cleanest and safest way to engineer a genome. Its most striking feature is that it allows the precise replacement of a sequence with another. For example, a deficient gene can be replaced with a functional copy in situ, without any modification elsewhere in the genome. Thus, the function is completely restored; it is not a mere compensatory effect, as is the case with random integration.

In classical homologous gene targeting, a fragment homologous to the locus to be modified is transfected in the cell. If one can select for insertion in the genome (with a selectable marker placed between flanking homologous DNA sequences), the majority of the insertion events in most organisms will be found at random locations all over the genome. A few events will result from the integration at the homologous locus by homologous recombination; however, one needs to screen extensively for such rare events.

This is not the case in a limited number of organisms and cell types where homologous integration is predominant: the yeast Saccharomyces cerevisae, the moss Physcomitrella patens, the avian DT40 lymphoid cell line, and a few modified or mutant Escherichia coli strains expressing recombination protein from the lambda phage or Rac prophage.

However, even in mammalian cells, homologous gene targeting can outnumber random integrations by orders of magnitudes when using MRS©: delivering a DSB in the targeted locus results in a very high frequency of homologous gene targeting. The frequency can reach a few percent of the cells, largely above the 10-5-10-6 found for random integration.

Site Directed Recombination

Site directed recombination consists in forcing recombination to occur at a defined location. Classical homologous gene targeting cannot be considered as site directed recombination for it implies only the selection of recombination at the desired locus among what is often a sea of non-targeted events.

In contrast, any process that would channel most recombination events in the targeted locus would be site directed recombination. Cellectis’ MRS© are ideal tools for site directed recombination: delivering a double-strand break in the locus targets the vast majority of recombination event to this locus, by homologous recombination.

Double-Strand Break Repair (DSBR)

http://www.cellectis.com/dsbr.html

Reverse Genetics

http://www.cellectis.com/reverse.html

meiosis

http://cancerweb.ncl.ac.uk/cgi-bin/omd?meiosis

A specialised form of nuclear division in which there two successive nuclear divisions (meiosis I and II) without any chromosome replication between them. Each division can be divided into 4 phases similar to those of mitosis pro, meta, ana and telophase). Meiosis reduces the starting number of 4n chromosomes in the parent cell to n in each of the 4 daughter cells. Each cell receives only one of each homologous chromosome pair, with the maternal and paternal chromosomes being distributed randomly between the cells. This is vital for the segregation of genes. During the prophase of meiosis I (classically divided into stages: Leptotene, Zygotene, Pachytene, Diplotene and Diakinesis), homologous chromosomes pair to form bivalents, thus allowing crossing over, the physical exchange of chromatid segments. This results in the recombination of genes. Meiosis occurs during the formation of gametes in animals, which are thus haploid and fertilization gives a diploid egg. In plants meiosis leads to the formation of the spore by the sporophyte generation.

 

Ulrich Melcher / recombination

In addition to the changes that genomes undergo due to mutations arising from unrepaired damage to DNA and replication errors, genomes change by a variety of other mechanisms, loosely classified as rearragements.

  • Two types of rearrangements can be distinguished mechanistically:
    • In site-specific recombination, specific target sequences on each of two DNA segments are sites of strand exchange, resulting in inversions, deletions and insertions.
    • In transposition, a specific target sequence on one DNA seqment is the site of initiation of transfer to another DNA sequence by one of several distinct mechanisms. Some transposition events require the progress of the replication fork.
  • Some rearrangements play important roles in the life cycle of the organism and are called programmed rearrangements.
  • Rearrangements are often called illegitimate recombination to distinguish them from general or homologous recombination in which exchanges occur between homologous DNA sequences.

One type of DNA rearrangement is catalyzed by site-specific recombinases.

  • Site-specific recombinases catalyze a reciprocal (inversely related) double-stranded DNA exchange between two DNA segments.
  • These recombinases recognize very specific sequences in both partners of the exchange.
    • The target DNA sequences can be on the same or on different strands.
    • If on different strands, an insertion, or integration, as occurs during lysogeny by lambda phage, results.
    • If on the same strand, the outcome depends on alignment of the target sequences.
      • In one alignment, inversion occurs. Examples include events catalyzed in the Flp and Hin inversion systems.
      • In the other alignment, deletion of a circular DNA from a linear DNA, or separation of a circular DNA into two circles results. These reactions occur in the resolution of cointegrates and the resolution of concatemers.

  • The site-specific recombinases catalyze the exchange by two different mechanisms: the Int-Flp and resolvase-invertase mechanisms.
  • Some site-specific recombinases are the sole proteins required for the exchange, but others require accessory factors for function. 
  • Though most described site-specific recombinases are from procaryotes, they are not limited to procaryotes. Procaryotic recombinases function in eucaryotes, providing a useful biotechnological tool.
  • DNAs of plant mitochondria have structures suggestive of site-specific recombination systems active in their formation.

Int-Flp Recombinases

  • The Flp recombinase belongs to one of two classes of proteins that catalyze inversions. Other members of the family catalyze phage DNA integration. The Int-Flp family includes lambda Int, Flp, and cre.
  • Three-dimensional structure studies provide insights into the mechanism used by the Flp recombinase to invert DNA sections.
  • Cre is the only protein required to catalyze inversion at lox target sites in inverting a segment of bacteriophage P. The lox spacer is 6 bp and 2 bp staggered cuts are made. The simplicity of the cre-lox system resulted in its adaptation in biotechnology to remove selectable marker genes from transgenic organisms to prepare them for commercial release. 
  • Lambda integrase requires several additional factors binding at sites surrounding the target "att" sites: xis, int, Hu and others.

http://opbs.okstate.edu/~melcher/MG/MGW3/MG3211.html

Mobio web book:

 http://www.web-books.com/MoBio/Free/Chap8.asp

Homologous recombination occurs between two homologous DNA molecules.  It is also called DNA crossover.  During meiosis, two homologous pairs of sister chromatids align side by side.  The DNA crossover is very likely to occur.  It could be as often as several times per meiosis.  

Figure 8-D-1.  DNA crossover.  (a) Two homologous pairs of sister chromatids align side by side.  (b) The two homologs are connected at a certain point  called chiasma(c) The two homologs exchange the DNA segment from the chiasma to the end of chromosomes.

Site-specific recombination occurs at a specific DNA sequence.  The first example was found in the integration between l DNA and E. coli DNA.  Both of them contain a sequence, 5'-TTTATAC-3', called the attachment site, which allows the two DNA molecules to attach together by base pairing.  Once attached, the enzyme integrase catalyzes two single strand breaks as in the Holliday model.  After a short branch migration, the integrase exerts a second strand cuts on two other strands.  Resolution of two Holliday junctions completes the integration process.

Figure 8-D-7.  Site-specific recombination between l DNA and E. coli DNA. 

DNA Recombination Technology

The procedure used in artificial DNA recombination is similar to the natural transpositional recombination.  The major difference is that researchers can choose any appropriate enzymes to cut the DNA molecules.  They are usually isolated from bacteria.  The role of these enzymes in bacteria is to "restrict" the invasion of foreign DNA by cutting it into pieces.  Hence, these enzymes are known as restriction enzymes.

 Table 9-A-1.  Commonly used restriction enzymes.

Note:  The "Recognition site" of a restriction enzyme is also called the restriction site.  In this column, the first line is from 5' to 3' and the second line is from 3' to 5'.  The arrow indicates the cleavage site.  If the cleavage site is not at the center, the restriction enzyme will generate sticky ends (web link) which can base-pair with other DNA fragments cleaved by the same restriction enzyme.   If the cleavage site is at the center, the restriction enzyme will generate blunt ends.

 


Illustrations from Genentech's Access Excellence

The action of a restriction enzyme

Inserting DNA into a plasmid 

Cloning into a plasmid

Cloning Vectorshttp://www.web-books.com/MoBio/Free/Ch9A4.htm

Technology - Homologous recombination

http://www.genoway.com/homologous_recombination.htm

When a DNA fragment is introduced in the nucleus of a cell it can integrate randomly into the genome. If the introduced DNA is identical (homologous) to a DNA sequence (locus A) present in the genome, then the integration can occur in the locus A. Based on that property it is then possible to target a mutation in a specific location of the genome.
This technology is called homologous recombination.
A foreign sequence (e.g., a Neo cassette) can be introduced by homologous recombination in a mouse gene A. The insertion of the foreign sequence is designed to prevent the transcription of the mouse gene A. The mouse gene A is no longer expressed; the gene A is "knocked-out". If the experiment is performed with mouse Embryonic Stem cells (ES cell), injection of the recombinant Embryonic Stem cells (ES cell) into blastocysts give rise to chimeras, heterozygous and homozygous animals, also called knock out mouse.
A transgene B (e.g., a sequence coding for a fluorescent protein) can be introduced by homologous recombination in a mouse gene A in such a way that the transgene B transcription is driven by the promoter of the mouse gene A. The transgene B is expressed with the same pattern of expression as the mouse gene A; this strategy is called "knock-in". If the transgene B is the human counterpart of the mouse gene A, then this strategy is called humanization and the mouse a humanized mouse.

 

Embryonic Stem cells http://www.genoway.com/es_cells.htm

Homologous Recombination/bio davidson

http://www.bio.davidson.edu/courses/genomics/method/homolrecomb.html

When an investigator wants to replace one allele with an engineered construct but not affect any other locus in the genome, then the method of choice is homologous recombination. To perform homolgous recombination, you must know the DNA sequence of the gene you want to replace (figure 1). With this information, it is possible to replace any gene with a DNA construct of your choosing. The method has a few more details than will be illustrated on this page, but the essential information is retained.

Figure 1. Diagram of gene targeted for replacement by an engineered construct. The coding sequence is illustrated by the box with flanking upstream and downstream DNA sequences provided. The arrows pointing away from the targeted gene represent the continuous chromosomal DNA.


The next step is to design and fabricate the DNA construct you want to insert into the chromosome in place of the wild-type allele. This construct may contain any DNA sequence of your choosing which means you can insert different alleles (both functional and non-functional ones), different genes or reporter genes (e.g. antibiotic resistance or green fluorescent protein). Regardless of what you want to insert, you must include some flanking DNA that is identical in sequence to the targeted locus (figure 2). In addition to the positive selection marker (e.g. antibiotic resistance) often a negative selection marker (e.g. thymidine kinase, tk) is added to the replacement vector. The negative marker is outside the region of sequence similarity between the vector and the targeted locus.

Fiugre 2. Diagram of engineered construct that will be used to replace the wild-type allele. The upstream and downstream flanking DNA sequences are identical to those which flank the targeted locus. The negative marker tk is shown in to the right of the region of sequence similarity.


The engineered construct is added to cells which contain the targeted gene of interest. By mechanisms that are poorly understood but are similar to what occurrs during meiosis and mitosis when homolgous chromosomes align along the metaphase plane, the engineered construct finds the targeted gene and recombination takes place within the homolgous (meaning identical in this case) sequences (figure 3). The recombination may take place anywhere within the flanking DNA sequences and the exact location is determined by the cells and not the investigators.

Figure 3. Diagram of alignment of DNA just prior to homolgous recombination. Amazingly, the DNA construct finds its way into the nucleus and then aligns itself with the targeted locus. The mechanism that performs this alignment is poorly understood but it does work better in some species than others. One of the best species for performing homologous recombination is yeast. Mouse is good mammalian system but homologous recombination does not work as efficeintly in human cells.


Once the cells have performed their part of the procedure, the end result is a new piece of DNA inserted into the chromosome. The rest of the genome is unaltered but the single targeted locus has been replaced with the engineered construct and some of its flanking DNA (figure 4). The original engineered construct has taken up the targeted gene of interest but since it cannot replicate in a nucleus, it is lost quickly in dividing cells while the modified chromosome replicates faithfully, including its new insert.

Cells that have undergone homologous recombination can be selected by addition of antibiotic to the growth medium (positive selection). Notice that the negative selection marker is not incorporated into the chromosome by homologous recombination.

Figure 4. Diagram of the final products of homologous recombination. The chromosome now contains a portion of the flanking DNA as well as all of the engineered construct which has taken the place of the original allele. The original allele has been recombined into the construct and thus is lost over time. From this point on, the cell will replicate the engineered construct as faithfully as any other portion of the chromosome.


If the targeting vector aligns in a non-homologous region of the genome, then recombination is random and the negative selection marker may become incorporated into the genome (figure 5).

Figure 5. Alignment of DNA just prior to non-homolgous recombination. In this case, the recombination will occur randomly. Notice that this time the tk marker will be included in the recombination event.


The final product of non-homologous recombination (figure 6) can survive positive (antibiotic) selection. However, there is a drug called gancyclovir that will kill any cell that contains the tk gene. So cells undergoing homologous recombination are grown in antibiotic to select for recombination and gancyclovir to kill any cells that successfully conducted non-homologous recombination.

Figure 6. End products of non-homolgous recombination. The positive and negative selection markers are incorporated into the chromosome so gancyclovir will kill cells with modified chromosomes as shown here.

Knockout Mouse

A knockout mouse has had both alleles of a particular gene replaced with an inactive allele. This is usually accomplished by using homologous recombination to replace one allele followed by two or more generations of selective breeding until an breeing pair are isolated that have both alleles of the targeted gene inactivaated or knocked out. Knock out mice allow investigators determine the role of a particular gene by observing the phenotype of individuals that lack the gene completely.

In the early 1990's a new method was developed to delete a specific portion of DNA. The procedure took advantage of the basic research performed on the bacteriophage called P1. In this virus, there is an enzyme called cre and particular DNA sequences called lox P sites. The lox P sites work in pairs and they flank a segment of DNA called a target (figure 1).

Figure 1. A pair of lox P sites (yellow ovals) flanking the target DNA (purple) to be deleted.

When the cre enzyme binds to the lox P sites, it cuts the lox sites in half and then splices together the two halves after the target DNA has been removed (figure 2).

Figure 2. After the cre enzyme has excised the target DNA, one lox P site is left behind and the two flanking fragments of DNA are spliced together. The target DNA is excised and degraded.

Molecular biologists recognized the specificity and utility of this viral recombination system and put it to good use. Now if you want to excise a piece of DNA at a particular time, all you need to do is to flank the target DNA with a pair of lox P sites and introduce the cre enzyme when you want the target excised. Mike Snyder's group used this to added epitope tags onto yeast proteins (Proteomics Chapter).

An additional twist is to express a Cre transgene under control of an inducible(produced because of stimulation by an inducer, formed by a cell in response to the presence of its substrate <inducible enzymes>) promoter so you can delete the target DNA inside selected cells of a transgenic organism when you want it deleted.

Epitope Tags for Antibody Bindinghttp://www.bio.davidson.edu/courses/genomics/method/Epitopetags.html

Expression Plasmids with Inducible Promoters http://www.bio.davidson.edu/courses/genomics/method/plasmid_inducible.html

Vectors:

http://dir.niehs.nih.gov/dirkoc/vectors.htm#conditional

Target Gene

Target Gene

 

Basic
This is a general knock out replacing exon 2 with a neomycin resistance cassette. This most basic of the systems works as well as any for a simple knock out.

Basic Knock Out

 

Lox-Neo
In this knock out we have flanked the Neomycin cassette with Lox P site. This will allow either in vitro or in vivo removal of the Neo cassette. This is to remove any potential effects of the neo promoter, which is generally a strong ‘bi-directional’ system.

Lox-Neo Knock Out

 

Lox71-Neo
Similar to the Lox-Neo construct, this vector has Lox sites flanking the neo cassette. The difference is that one of the sites is a mutated Lox P, a Lox 71. This mutation will allow for the insertion is a plasmid in the future to either replace the removed exon with a modified version (see Knock In) or any other DNA you choose.

Lox71-Neo Knock OutPage Top

 

Conditional Knock Out
In this most useful of knock outs, the exon of choice is flanked by the Lox P sites, This will allow the excision of the inclusive region by use of any of hundreds of tissue and stage specific Cre transgenes. A great choice when dealing with lethal knockouts and broad ranging phenotypes. The Neo cassette in this construct is flanked by FRT sites, this will allow for the removal of the neo, with out affecting the rest of the gene.

Conditional Knock Out

 

Reporter Knock Out
This ‘two tools in one’ approach gives the investigator both a basic knock out, and lacZ reporter knock in. Having the same effect of removing the exon of choice, it will act as a knock out mouse, but the addition of the LacZ (or any other) reporter gene will reveal the expression pattern of the gene of interest.

Reporter Knock out

 

Knock In
The Knock in could prove to be one of the more powerful systems we have. Two of the more interesting uses would be the introduction of point (or small) mutations, and the humanized mouse model. The introduction of the point mutation can generate either a complete loss of function, or a partial loss of function. In the humanized model, genes or exons can be replaced with their human equivalent when there seems to be a functional difference.

Knock In Page Top

CONDITIONAL CONTROL OF GENE EXPRESSION IN THE MOUSE

Mark Lewandoski    about the author

Preface

One of the most powerful tools that the molecular biology revolution has given us is the ability to turn genes on and off at our discretion. In the mouse, this has been accomplished by using binary systems in which gene expression is dependent on the interaction of two components, resulting in either transcriptional transactivation or DNA recombination. During recent years, these systems have been used to analyse complex and multi-staged biological processes, such as embryogenesis and cancer, with unprecedented precision. Here, I review these systems and discuss certain studies that exemplify the advantages and limitations of each system.

Summary


Temporal and spatial control of gene expression in the mouse can be achieved using binary transgenic systems, in which gene expression is controlled by the interaction of an effector protein product on a target transgene. These interactions are controlled by crossing mouse lines, or by adding or removing an exogenous inducer.
Binary transgenic systems fall into two categories. One is based on transcriptional transactivation and is well suited for activating transgenes in gain-of-function experiments. The other is based on site-specific DNA recombination and can be used to activate transgenes or to generate tissue-specific gene knockouts and cell-lineage markers.
The most commonly used transcriptional systems are based on the tetracycline resistance operon of Escherichia coli. The effectors of these systems fall into two categories defined by whether transcription activation occurs upon the administration or depletion of a tetracycline compound (usually doxycycline).
The Gal4-based system is a transactivation system that does not require an inducer, but Gal4 transcriptional activation can be controlled by synthetic steroids when a mutated ligand-binding domain is incorporated into a Gal4 chimeric transactivator.
The most widely used site-specific DNA recombination system uses the Cre recombinase from bacteriophage P1. The Flp recombinase from Saccharomyces cerevisiae has also been adapted for use in mice.
By using gene-targeting techniques to produce mice with modified endogenous genes that can be acted on by Cre or Flp recombinases expressed under the control of tissue-specific promoters, site-specific recombination can be used to inactivate endogenous genes in a spatially controlled manner.
Cre/Flp activity can also be controlled temporally by delivering cre/FLP-encoding transgenes in viral vectors, by administering exogenous steroids to mice that carry a chimeric transgene consisting of the cre gene fused to a mutated ligand-binding domain, or by using transcriptional transactivation to control cre/FLP expression.
The irreversibility of site-specific recombination makes this technique uniquely suited for a new type of analysis in which the transient tissue-specific expression of cre/FLP is used to permanently activate a reporter target gene for cell-lineage studies.

site specific recombination
definition:A crossover event, such as the integration of phage lambda, that requires homology of only a very short region and uses an enzyme specific for that recombination. Recombination occurring between two specific sequences that need not be homologous; mediated by a specific recombination system.

Site-Specific Recombination

http://www.mun.ca/biochem/courses/3107/Lectures/Topics/Site_specific_Recomb.html

Site-specific recombination differs from general recombination in that short specific sequences which are required for the recombination, are the only sites at which recombination occurs. These reactions invariably require specialized proteins to recognize these sites and to catalyze the recombination reaction at these sites. The steps and features of the general recombination reaction, however, still apply:

  • strand exchange
  • formation of a Holliday intermediate
  • branch migration
  • resolution.

Because they involve specific sites, there are really only two types of site-specific recombination reaction

Inverted repeats

If the two sites at which recombination will take place are oriented oppositely to one another in the same DNA molecule then the following illustrates the sequence of events that will take place:

The net result is that the segment of DNA between the two recombinogenic sites has inverted with respect to the rest of the DNA molecule.

In other words, recombination at inverted repeats causes an inversion.

 

Direct repeats

If the two sites at which recombination will take place are oriented in the same direction in the same DNA molecule then the following illustrates the sequence of events:

 

The net result is that the segment of DNA between the two recombinogenic sites has been deleted from the rest of the DNA molecule and appears as a circular molecule.

In other words, recombination at direct repeats causes a deletion.

Note that the reverse reaction -- the recombination of a circular molecule with another DNA molecule (either circular or linear), brings about a fusion of both molecules or the integration of one molecule into the other. The integrated segment will be flanked by directly repeating sequences which can, of course, be used to excise the integrated segment again.

[S32-20]

Site-specific recombination reactions provide an unusual but important mechanism for regulating gene expression. Since the order of the genes in an organism will change as a result of site-specific recombination, the affected genes can be kept in a silent state until after the recombination has taken place. This type of control occurs in the regulation of gene expression in a differentiating cell.

There are two major groups of enzymes that carry out site-specific recombination reactions; one group - known as the tyrosine recombinase family - consists of over 140 proteins. These proteins are 300-400 amino acids in size, they contain two conserved structural domains, and they carry out recombination reactions using a common mechanism involving a the formation of a covalent bond with an active site tyrosine residue. The strand exchange reaction involves staggered cuts that are 6 to 8 bp apart within the recognition sequence. All of the strand cleavage and re-joining reactions proceed through a series of transesterification reactions like those mediated by type I topoisomerases.

The Int protein is the best known and studied member of this family. Others are the XerD protein of E. coli (which is responsible for separating chromosomes at the end of replication), the Cre protein of bacteriophage P1, and the FLP protein of yeast.

Using General Recombination in a Site-specific Event

The construction of interposon mutants

Whenever one is studying any particular gene, one invariably wants to know what happens if the gene cannot be expressed in vivo. To do this, one could use site-specific mutagenesis to place very specific base pair changes within a coding region. However, this is both difficult and requires a lot of information about the system to know exactly what sort of chnages must be made.

A much easier way to do this is simply to insert a large piece of DNA in the middle of the gene. This should block expression of the gene since transcription is unlikely to proceed all the way through the inserted DNA, and, even if it did, the mRNA that is made would not be translated into a functional protein.

If you're going to do this, then you need a way to know that the piece of DNA which you wish to insert really has been inserted. If the piece of DNA to be inserted codes for an antibiotic resistance, then you can select for its presence quite easily.

The following diagrams outline the procedure that is followed:

Cloning the antibiotic resistance piece of DNA into the middle of his gene was straightforward and only involved standard cloning techniques.

 

 

If we now transform or conjugate the plasmid into the bacterium of choice, at this point we have two copies of the target gene in the cell, but

  • one of them has an antibiotic resistance element in the middle of it, and

  • if, we are studying an organism other than E. coli, it is on a plasmid that cannot replicate.

(Note that it is not always possible to transform bacteria with DNA. If transformation is not possible then conjugation or electroporation can be used to get DNA into the bacterium.)

Now if we select for cells that grow on media containing the antibiotic, they cannot do so unless the antibiotic resistance gene is maintained in the cell. However, if it is located on a plasmid that cannot replicate, it must integrate into the chromosome in order to be maintained and expressed.

This can only happen if there is a region of homology in the plasmid which can recombine with the host chromosome.

There are, in fact, two such regions - one on either side of the antibiotic resistance gene, i.e. the two halves of the original target gene.

A single recombination on one side only will result in the integration of the whole plasmid containing the interrupted target gene. This would leave us with two copies of the RNA-binding protein gene in the cell:

 

Recombination on both sides would get rid of the wild-type copy of the gene. However, this can be difficult to find unless specially designed plasmid vectors are used that force the second recombination event to take place.

 

So this Interposon mutagenesis requires a site-specific recombination event in the sense that the recombination events must occur only in the range of places determined by a cloned piece of DNA. However, the mechanism by which this recombination takes place is still general homologous recombination.

 

Site-Specific Recombination

Alignment of sites for homologous recombination occurs via DNA - DNA (base-pairing) interactions. Another important class of recombination reactions is called site-specific recombination. It is directed by highly specific DNA - protein interactions, although a short stretch of DNA homology occurs at the actual site of cutting and resealing. Information about site-specific recombination is most advanced for the mechanism by which phages, such as , become integrated at specific sites on an infected bacterium's chromosome.

The chromosome integrates at a specific site on the E. coli chromosome, attB, which maps between genes involved in galactose utilization and biotin synthesis (the gal and bio markers), as illustrated in Figure 25.17. Integration occurs at a specific site on the phage chromosome called attP.

Two proteins are required for this site-specific recombination. They are as follows:

1. Phage integrase (Int) - the product of the int gene; and

2. Integration Host Factor (IHF)--an E. coli protein.

Phage DNA must be supercoiled for the recombination to occur. Supercoiling, plus distortion created by Ihf binding to specific sites in attP, facilitates Int binding at adjacent sites. The nucleoprotein structure is called an intasome, which aligns with attB (also bound with Int). At the core of the and E. coli sequences is a 15-base region of complete homology (Figure 25.31). In each of these sequences, Int creates a staggered cut, with a 7-nucleotide overlap. The ends then exchange to form a Holliday junction and a DNA ligase activity of Int joins the ends covalently.

Upon integration, is dormant. Later, changes in the cell activate the virus which excises from the host genome by a reversal of the above steps to yield a circular virus. In this reaction, a protein called Xis is required in addition to Int and IHF.

Site Specific Recombination

Gene bridges products:

FLPe
Site-Specific Recombination mediated by site-specific recombinases (SSRs, like Cre and FLP) are useful tools for genomic engineering of eukaryotic cell lines, animals and plants. A range of applications has been demonstrated, including chromosomal translocations and large deletions, tissue-specific and conditional knockouts, inducible gene expression and site-specific integration and precise removal of selectable markers.
Plasmids and Templates for Cre applications
The  plasmids and templates listed exert functional cassettes like selection cassettes  flanked by loxP-sites designed for the use in Eu- or Prokaryotes. They can be used to generate targeting constructs in mammals, or any other DNA construct meant to use Cre-Recombinase. 
Plasmids and Templates for Flp applications
The  plasmids and templates listed exert functional cassettes like selection cassettes  flanked by FRT-sites designed for the use in Eu- or Prokaryotes. They can be used to generate targeting constructs in mammals, or any other DNA construct meant to use either Flp- or Flpe-Recombinase.
Strains
These bacteria can be used as deleter strains. Either you would like to check the functionality of your DNA construct harboring loxP- or FRT-sites or you would like to excise FRT- or loxP-flanked DNA stretches during a modification task we provide a simple solution.

references http://www.genebridges.com/web/technology/ssrecombination.htm

Red/ET Recombination can be applied to numerous DNA engineering tasks

 

Site specific recombination

http://www.brunel.ac.uk/depts/bio/project/molgen/bacteria/recomb/sitespec.htm

In site specific recombination a DNA molecule is cut in both strands at two specific positions, and the ends are rejoined to new partners in a series of catalytic steps which do not involve any synthesis or degradation of DNA as opposed to the mechanisms of generalized recombination. For the reaction to occur relatively short pieces of homologous DNA (a few tens of basepairs) must be present both on the incoming and the resident DNA molecule; this process is mediated by socalled recombinases which are further subdivided into subtypes called integrases and resolvases.

Examples for integrase activity include the integration and excision of bacteriophage lambda (mediated by Int and Int/Xis respectively), the monomerization of plasmid oligomers (E.coli XerC) as they occur during plasmid DNA replication, recombination of the linear P1 DNA into a circular molecule (P1 Cre), and the Flip/Flop rearrangements on the 2micron circle of S. cerevisiae (2 micron FLP).

Examples for resolvase activity include transposon-resolvases (Tn3, gamma/delta, Tn21, Tn552)and gene inversion events mediated by socalled invertases such as Hin (catalysing inversion of its own gene and by doing so triggering a switch from one flagellin type to another one in S. typhimurium) and Gin (inversion of the G-fragment in bacteriophage Mu) and Cin (bacteriophage P1).

Individual steps in site-specific recombination

In contrast to homologous recombination where basepairing leads to the alignment of homologus sequences, it is DNA-binding proteins which play the primary role in site-specific recombination in bringing the two DNA-molecules together. Theses proteins bind to identical short recognition sequences which are usually (but not necessarily) present on both interacting DNA molecules; for example the integration sites of bacteriophage lambda (attP) and on the E.coli chromosome (attB) do not share any sequence homology.

The binding of those specific proteins then triggers the formation of a synapsis between the two DNA strands,mediated by protein/protein and/or protein/DNA interaction followed by DNA-bending with the aid of accessory proteins such as IHF, FIS or HU.

In the reaction of resolvase-type recombinases the next step involves the 'staggered' cleavage of DNA within the short crossover-sites which have imperfect dyad symmetry and hence bind two molecules of recombinases per DNA molecule ie four in total thereby generating a 2bp overhang. The recombination reaction itself is initiated by a nucleophilic attack of a conserved serin in the active site of the enzyme towards the 5'-end of the cut phospho-diester backbone.

In the integrase family of recombinases a 6-8 bp staggered cut is made and the 3'-ends of the DNA at the cleavage site are joined to a hydroxyl-group of a conserved tyrosine residue in the active site.

Note therefore that in both instances one strand of the cut DNA molecule is covalently joined to the recombinase via a activated phospho-ester bond thereby in effect conserving the energy originally retained in the sugar-phosphate backbone.

The next step involves strand exchange which, in integrases, occurs in two steps via the formation of a socalled 'Holliday junction', a intermediate also found in honologous recombination between two double stranded DNA molecules.

Conclusion

Site specific recombination events are widespread and lead to a diverse range of structural DNA rearrangements which may have major effects on cell physiology by altering for example the regulation of gene expression. This is achieved by the placing of promoters near the termini of the DNA segment to be inverted; since promoters act in a polar fashion the respective gene is only switched on when the DNA segment is placed in one particular orientation

Title: Site-Specific Cassette Exchange and Germline Transmission with Mouse Es Cells Expressing Phic31 Integrase 2003
 
Interpretive Summary: This article describes the use of the Streptomyces phage phiC site-specific recombination system through a DNA exchange event to insert DNA precisely into mouse stem cells. The inserted DNA led to germline transmission of the introduced transgene. This exemplifies the use of phiC31 site-specific integration to engineer mammalian cells that could eventually lead to the precise engineering of farm animals.

Technical Abstract: Currently two site-specific recombinases are available for ngineering the mouse genome: Cre from P1 phageand Flp from yeast. Both enzymes catalyze recombination between two 34'base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.

SITE-SPECIFIC RECOMBINATION

 http://www.bio.brandeis.edu/classes/biol122a/

A.  Introduction: 

      1. Homologous recombination vs. site-specific recombination:

            Homologous recombination occurs between DNA with extensive sequence homology anywhere within the homology. Site-specific recombination occurs between DNA with no extensive homology (although very short regions may be critical) only at special sites. The protein machinery for the two types of recombination differs too.  Strand exchange during site-specific recombination occurs by precise break/join events and does not involve any DNA loss or DNA resynthesis.

      2. Physical consequences of site-specific recombination events:

            a. Intermolecular reaction of circle + circle = integration

            b. Intramolecular reaction between two sites on circle with direct orientation = excision or resolution.

            c. Intramolecular reaction between two sites on circle with inverted orientation = inversion ("flipping").

      3.  Biological consequences of site-specific recombination events

            a.  Prophage integration/excision systems. Temperate bacteriophage establish lysogeny by integration of its genome (in a repressed state) into a special site on the bacterial chromosome by recombination at the attachment (or att) site.  Bacteriophage lambda integration excision is the best-characterized site-specific recombination system.  Other phage which integrate/excise include: P2, j80, P22 and 186. 

            b. Resolution/excision systems. 

                  1. Transposon cointegrate resolution.  Some transposons move by a replicative process that results in a "cointegrate" intermediate containing two copies of the transposon.  Site-specific recombination then occurs between the two copies of the transposons.  (More about this when we talk about transposition.) Transposons of this type include Tn3, gamma-delta, Tn21, Tn501 and Tn1721. 

                  2. Replicon dimer resolution. Homologous recombination between circular chromosomes or plasmids yields dimers, trimers, tetramers, etc. These larger structures do not segregate as well as the starting monomer plasmid. Therefore, some plasmids encode site-specific recombination systems to produce monomers from multimers.  Examples are the cer system of ColE1 plasmid and the cre/lox system of bacteriophage P1 which, in the lysogenic state, replicates as a plasmid. E. coli has a site-specific resolution site near the terminus of replication called “dif” at which the XerCD proteins act to resolve chromosome dimers to monomers before cell division. 

                  3. Developmental excision.  Bacillus subtilis undergoes a site-specific recombination event during sporulation to assemble a transcription factor specific for sporulation functions. The development of Anabena, a cyanobacterium, into the nitrogen-fixing heterocyst form involves two intramolecular site-specific recombination events to activate nitrogen fixation genes. 

            c. Inversion systems. 

                  1. Antigen switching/host range.  Site-specific inversion results in the expression of one of two forms.  Important mechanism for the generation of diversity within a population. Strategy is to avoid a mounting immune response against a particular antigen (in the case of Hin and Fim) or to prevent depletion of host, in case of Gin, Cin.  Inversion is reversible.  Examples: Hin: switching of Salmonella flagella antigen forms (stands for H inversion, H being flagellar genes).  Fim: switching of pilin antigens ("fimbriae") of E. coli. Gin, Cin: switching of host range of bacteriophage Mu and P1, respectively. Pin: function unknown, cryptic e14 prophage in E. coli. The Hin, Gin, Pin and Cin invertase proteins are highly homologous in amino acid sequence. 

                  2. FLP: The 2-micron plasmid of the yeast Saccharomyces cerevisiae inverts a segment.  This changes the relative orientation of the replication forks, promoting the amplification of the plasmid to high copy number. 

2.  The lambda Int system. 

      a. attP (the phage site) + attB (the bacterial site) results in the integration of lambda into the E. coli chromosome.  Integrated lambda DNA is flanked by two hybrid att sites:  attL (left) and attR (right)--each is derived from one half of attP and attB.  This process requires the lambda Int protein and the bacterial protein  IHF ("integration host factor"). IHF is a heterodimer of two E. coli proteins. 

      b. attL + attR results in the excision of lambda (restoring attP and attB) during the induction of lambda prophage. This process requires Int, IHF, and lambda Xis ( for "excise") proteins. E. coli Fis protein stimulates the excision but is not required absolutely. 

      c.  All att sites consist of a homologous 15 bp"core" region where the exchange takes place.  Flanking the core are two Int binding sites in inverted orientation.  The attP site is large, having 235 bp with complex series of binding sites for Xis, Int, IHF and Fis on either side of the core.  The attB is much simpler at 30 bp in size and consists only of the core and the two core Int binding sites. 

3. Mechanism of site-specific recombination integration: lambda Int. 

      a. The intasome. Specific proteins bind to the attP site forming a highly ordered nucleoprotein structure, wrapping 230 or so bp of DNA around a protein complex.  IHF induces sharp bend in the DNA of 140° when it binds  This bending is thought to be important for assembling the intasome complex. Int binds DNA both at the "core" region and at the two "arm" regions. These two modes of binding are different: different DNA sequences are recognized and the sites do not compete with each other. This bivalent (literally "double-strength") nature of Int binding is thought to allow subsequent interaction of the intasome and attB.  Binding is facilitated by superhelicity of attP DNA.  There are both cooperative and competitive interactions between binding proteins. These are thought be important for regulating and determining the directionality of the integration vs. the excision reaction. 

      b.  Synapsis. This part of the recombination process where the two molecules come together is not completely understood. The attB DNA is captured by the intasome complex.  Core homology is not required and synapsis must therefore be facilitated by protein interactions. 

      c.  Strand exchange.  

            1. Nicking occurs on each strand producing a 7 bp staggered cut.  The DNA is broken with the region of sequence identity of attP and attB. 

            2. No ATP or high-energy cofactor is required. The energy for the reaction derives from the cleavage of the phosphodiester bond of DNA, which is conserved via the formation of a transient enzyme-P-DNA linkage. DNA is joined via a 3' phosphate to a tyrosine residue of Int. 

            3. The phosphate is then transferred to the analogous strand of the second parental molecule, generating recombinant molecules with no nicks or gaps.  (This contrasts with homologous recombination where some post-recombination DNA repair synthesis and subsequent DNA ligation must occur. )  

            4.  Evidence suggests that the two strands are exchanged sequentially. That is, first the leftward strands are exchanged, then the rightward strands are exchanged. The first strand exchange, which results in the formation of a Holliday junction (crossed-strand structure), can occur even when there is no homology in the core regions between the two parental molecules. The first strand exchange is a reversible reaction, the second is not. 

4.  Regulation and directionality of lambda integration/excision. 

      a. Although you may think that excision is formally the reverse reaction of integration, the two recombination reactions are different mechanistically. Excision is an intramolecular reaction; integration an intermolecular recombination reaction. attL and attR are different in size and binding properties than attP and attB, because of the rearrangement of sequences.  The directionality of integration vs. excision is determined by the relative levels of the binding proteins in a complex way. Xis inhibits integration and promotes excision.  Although IHF is required for excision, binding of IHF to second site inhibits excision; Fis stimulates excision and has no effect on integration.

      b. These IHF and Fis effects allow directionality to be determined by growth phase of the host cell: IHF is high in stationary phase, Fis is high in log phase. The strategy here is that if the phage will be unable to replicate due to lack of growth nutrients delay excision (stationary phase: IHF high = inhibition of excision, Fis low = inhibition of excision).

      c. Under high Int/Xis conditions, these IHF/Fis effects are overridden.  Cooperative binding of these proteins allows more efficient excision.  High Int/Xis levels might be expected after induction of the SOS response, meaning strategically that if the cell is in trouble, lambda should get out while it can no matter what the growth phase.  Conversely, low Int/Xis can result from co-infection with other lambda  phage.  The strategy in this case is to not excise when competitors for replication are present, so the system become more sensitive to growth phase of the cell.

      d. In summary, the complex competitive and cooperative binding at the att sites allows the direction of phage recombination reactions to be adjusted to both host physiology and competing bacteriophage.

5. Use of site-specific recombination in genetic analysis

      a. High efficiency integration: The cre/lox system of bacteriophage P1 is being used as a simple integration system since the lox site is relatively small and recombination requires only the Cre protein and no other accessory factors. Circular segments of DNA containing a lox site can be transfected into cells and efficiently integrated at a resident lox site in the chromosome. 

      b.  Conditional expression. It is difficult to analyze the function of essential genes. In higher eukaryotes, if a gene is essential for early development, effects on later processes cannot be ascertained. Site specific recombination can be used to delete a function at a particular stage by flanking the gene by direct lox sites and by inducing expression of Cre with a regulatable promoter such as a heat-shock promoter. Cre will catalyze excision and deletion of the gene and the phenotype in the resultant organism can be assayed.

      c.  Mitotic recombination. A heterozygote for a particular mutation can be made and mitotic recombination that results in a homozygous mutant state can be induced in a subset of cells of the organism. A site-specific recombination site is placed centromere-proximal to the heterozygosity on the homologous chromosomes and expression of the site-specific recombination protein induced. Recombination between homologs will result in homozygous wild-type and homozygous mutant sectors. FLP and FRT (protein and sites of the site-specific recombination system of yeast 2 micron circle plasmid) have been used in this way in Drosophila

DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation