How does Homologous recombination work?

Question

DNA repair, NHEJ

Answer 1

Homologous Recombination

1.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 homologous recombination, you must know the DNA sequence of the gene you want to replace. 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.

2.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. 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.

3.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. The recombination may take place anywhere within the flanking DNA sequences and the exact location is determined by the cells and not the investigators.

4.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. 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.

5.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.

6.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.

7.The final product of non-homologous recombination 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.

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