Rapid generation of long synthetic centromeric tandem repeats for mammalian artificial chromosome formation

摘要

Methods are described for construction of long synthetic arrays of DNA repeats, such as alphoid repeats or other repeat sequences. The methods include concatamerization of DNA into short repeats (for instance using rolling circle amplification or directional in vitro ligation), followed by assembling the short repeats into long arrays by homologous recombination during transformation into microbe cells. These methods can be described generally as Recombinational Amplification of Repeats (RAR). The long arrays are engineered centromere-like regions that allow one to construct mammalian artificial chromosomes with a predefined centromeric region structure. Artificial chromosomes, including human artificial chromosomes with a regulated centromere, and methods of their use are also provided.

主权项

1. A method of generating a mammalian artificial chromosome (MAC) having a regulated centromere, comprising:
assembling one or more engineered centromeric sequences into a vector, wherein the engineered centromeric sequence comprises (1) at least one mammalian repeat sequence comprising an alphoid repeat sequence, a mouse minor satellite sequence or a synthetic sequence at least 90% identical to such a repeat sequence; and (2) a tet Operator (tetO) sequence wherein binding of a transactivator to the tetO sequence disrupts the function of the centromere of the MAC.