It has been a long time:) But GGS is back:)
Today we are going to look at Gene Targeting Method.
Are you ready?? Let's go!
Method: Gene Targeting
About: Simply saying, gene targeting is a method that allows to remove/alter a specific DNA sequence from the genome of cell.
What: Knockout of exemplary gene A in order to study its function.
How: By generation of a targeting vectors and their subsequent application.
Before we start, we need to know a little bit more about gene targeting, so:
Gene targeting - is a multistep process that allows to delete or mutate a gen of interest. Gene targeting can be either permanent or conditional. In the former a specific DNA sequence is deleted, where in the latter such sequence is retained but can be deactivated upon specific conditions.
Ok, lets start the fun. We are going to split the process into few steps:
1. mapping of genomic locus of exemplary gene (will called it gene A, from now on),
2. design the targeting strategy,
3. and finally screening and detection of knockout bearing cells.
1. Mapping of gene A genomic locus. To disrupt gene A we need to gather a genomic DNA sequence bearing the locus of interest. To map the genomic locus of gene A we can use either its cDNA (complementary DNA which is simply just a coding sequence of the gene) or mRNA sequence. If such information is not available in database (for example NCBI database) it has to be first obtained by cloning gene A and its sequencing. But let's imagine that a coding sequence of gene A is known. We use such sequence to screen the genomic database by performing BLAST search against database (see picture below).
Cartoon above represents a genomic locus of an exemplary gene A, aligned with its cDNA. The roman and arabic numbers show exons (coding sequence) and introns, respectively (notice that cDNA do not contain introns:). As you can see by performing such database search (BLAST) we are able to map a genomic localization of specific gene. The real BLAST result is shown on the figure below.
You can see here two exemplary bits of the cDNA sequence aligned against the genomic DNA. Notice that Query and Subject sequences are cDNA and genomic locus, respectively. The top one corresponds to the first exon (I) and the bottom to the last exon (V). Such search gives us two information:
- genomic localization of gene A (top table, gene A is present on chromosome 2),
- chromosomal position of gene A - notice that top bit, an exon I (Query position 1) is located at a genomic position 5834742 (this is were our coding sequence starts in the genome). The same for the exon V, which ends at genomic position 5839788. We know that there is 5 exons in total, so we can estimate the size of gene A locus which is:
Size of gene A genomic locus = 5839788 - 5834742 = 5046bp = 5.05kb
In this case our genomic locus is relatively small and we will be able to knockout whole sequence of gene A. Some gene loci are longer and different approaches might applied for their disruption (I am working with 122kb locus which is around 24 times more than our exemplary gene A:). In our study case we will try to knockout complete sequence of gene A.
2. Design of targeting strategy. Strategy includes:
- desing of targeting vector (this will be used to disrupt gene A),
- desing of probes, (these will be used to detect knockout cells).
Notice also that three HindIII restriction sites are present in the DNA region of interest. Two outside the gene A (up- and downstream to gene A) and one inside the gene A sequence. When gene A will be replaced by restriction cassette, the central HindIII site will not be present anymore. In this way a digestion pattern of genomic DNA will change. If locus is not targeted it will show wild type like pattern; 5'probe will detect 5.5kb band and 3'probe a 6.5kb one. But when targeting event takes place both probes should recognize a band of 11.0kb (that depends on restriction cassette size). To detect such change Southern Blot will be used.
Notice also that 5' and 3' probes will hybridize to DNA regions outside arms (upstream to 5' arm for 5' probe and downstream to 3' arm for 3' probe). It is important that probes are localized in the region outside arms. This is because in some cases our targeting vectors will be randomly integrated into genomic DNA (will not target gene A). If probes would hybridize to an arm region, they would pick up also those random integration events which are not of interest. This way we make our detection step easier
3. Screening and detection of knockout cells. When strategy is ready and targeting vectors are generated, we put them into action:). Wild type cells are transfected with a targeting vector and usually left around 24h to let them recover from stress. Next day cells are treated with a specific drug, depending on the cassette used and plated into 96 well plate. Conditions here are specifically optimized such approximatelly one cell that uptaken the DNA should be present in each well. This, and only this cell will grow and give a rise to a colony (other cells should die as they are not resistant to the drug). After few days single colonies are picked and expanded, so we have enough cells to extract DNA and freeze other half of cells for the time of screening. In the next step DNA from each clone (usually from 20-100 clones per transfection) is isolated and digested with an restriction enzyme (in our case HindIII). For the Southern Blot technique tutorial click here. An exemplary result is shown on the picture below.
As you can see clones 1, 2, 3, 4, 5, 6 and 8 are targeted as the digest pattern of genomic DNA has changed comparing to WT lane and non-targeted C7 clone. In this case 5'probe was used. We observe a correct band shift from 5.5kb to 11.0 kb. Notice also that there still is a WT band in each of the targeted clones. This is because there are two copies of gene A in each cell (there are usually two alleles of a gene). Clones 1, 2, 3, 4, 5, 6 and 8 are called heterozygotes for gene A (one targeted allele one WT allele). To obtain a full knockout we need to perform another round of a targeting on one of those heterozygotes using another targeting vector (bearing different resistance cassette, we cannot use the same vector because heterozygotes are already resistant to the drug, so we would not be able to select against the knockout cells). In this case wild type band will dissapear and only targeted band will be present.
I hope you enjoyed it:)
CyA Soon
Maciek GGS
3. Screening and detection of knockout cells. When strategy is ready and targeting vectors are generated, we put them into action:). Wild type cells are transfected with a targeting vector and usually left around 24h to let them recover from stress. Next day cells are treated with a specific drug, depending on the cassette used and plated into 96 well plate. Conditions here are specifically optimized such approximatelly one cell that uptaken the DNA should be present in each well. This, and only this cell will grow and give a rise to a colony (other cells should die as they are not resistant to the drug). After few days single colonies are picked and expanded, so we have enough cells to extract DNA and freeze other half of cells for the time of screening. In the next step DNA from each clone (usually from 20-100 clones per transfection) is isolated and digested with an restriction enzyme (in our case HindIII). For the Southern Blot technique tutorial click here. An exemplary result is shown on the picture below.
As you can see clones 1, 2, 3, 4, 5, 6 and 8 are targeted as the digest pattern of genomic DNA has changed comparing to WT lane and non-targeted C7 clone. In this case 5'probe was used. We observe a correct band shift from 5.5kb to 11.0 kb. Notice also that there still is a WT band in each of the targeted clones. This is because there are two copies of gene A in each cell (there are usually two alleles of a gene). Clones 1, 2, 3, 4, 5, 6 and 8 are called heterozygotes for gene A (one targeted allele one WT allele). To obtain a full knockout we need to perform another round of a targeting on one of those heterozygotes using another targeting vector (bearing different resistance cassette, we cannot use the same vector because heterozygotes are already resistant to the drug, so we would not be able to select against the knockout cells). In this case wild type band will dissapear and only targeted band will be present.
I hope you enjoyed it:)
CyA Soon
Maciek GGS
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