Chromosomal Manipulation by Site-Specific Recombinases and Fluorescent Protein-Based Vectors

Abstract

Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.

Advances in New Technology for Targeted Modification of Plant Genomes

“…Cells in which the EXCH vector integrated at random or did not complete RMCE do not survive the selection. Alternative designs allow this same strategy to be used for repetitive gene stacking Uemura et al 2010 ;Van Deursen et al 1995 ). The deletion/duplication events result from non-sister chromatid recombination that generates a set of balanced and unbalanced chromatids; cells with the latter will not survive.…”

“…With the age of genomics upon us, the use of designed "balancer chromosomes" could be a critical element in understanding how genomic arrangements infl uence gene expression, metabolic pathway function, and pathway interactions on a global scale. However, inverted lox sites can also lead to unequal recombination between sister chromatids, generating dicentric and acentric chromosomes that are lost during subsequent cell divisions, precluding cell survival (Lewandoski and Martin 1997 ;Uemura et al 2010 ).…”

Recombinase technology: applications and possibilities

“…As orientation of the loxP sites will determine the type of recombination event observed, and since eukaryotes are diploid, complications can occur. With two loxP sites located on a single chromosome in directly repeated orientation a deletion event is expected, but duplication events have also been detected (Medberry et al 1995 ; Ramierez-Solis et al 1995 ; van Deursen et al 1995 ; Uemura et al 2010 ). The deletion/duplication events result from a non-sister chromatid recombination event that generates a series of balanced and unbalanced chromatids.…”

“…This form of chromosomal rearrangement can be used to study genetic abnormalities and establish balanced lethal systems to facilitate stock maintenance (Zheng et al 1999 ). This loxP orientation can also lead to unequal recombination between sister chromatids, generating dicentric and acentric chromosomes (Uemura et al 2010 ). Chromosomes in these configurations are lost during the next cell division, generating monosomic cells (Lewandoski and Martin 1997 ; Uemura et al 2010 ).…”

Multiple expression cassette exchange via TP901‐1, R4, and Bxb1 integrase systems on a mouse artificial chromosome

“…These reactions are designed using site‐specific recombinase (SSR) systems. Cre‐loxP from bacteriophage P1 and Flp‐FRT from Saccharomyces cerevisiae are the most widely used systems. These Cre or Flp recombinases recognize specific nucleotide sequences (loxP or FRT), and perform a reversible catalytic reaction between two loxP or FRT sites, respectively.…”