Genetic microsurgery by laser: establishment of a clonal population of rat kangaroo cells (PTK2) with a directed deficiency in a chromosomal nucleolar organizer

Abstract

Abstract. An ultraviolet laser beam was focused to a submicron spot on one of the nucleolar organizer regions of mitotic chromosomes of rat kangaroo cells in tissue culture. The daughter cells were isolated and cloned into a viable population that maintained the directed nucteolar deficiency. It is concluded that the laser can be used to delete preselected genetic regions and the genetic deletion is maintained as a heritable deficiency in subsequent daughter cells.

Gene inactivation by multiphoton-targeted photochemistry

“…2). The cells had small dark nuclear bodies that are typical of cells whose ribosomal DNA have been inactivated (6,7,9). The double-nucleolar prophase cells having one nucleolus and associated chromosomes irradiated produced daughter cells with one nucleolus each (Fig.…”

“…Earlier single photon experiments with low power visible and UV lasers, as well as high power multiphoton microplasma-inducing laser beams, demonstrated that irradiation of the nucleolus and the adjacent attached chromosomes results in daughter cells with a corresponding reduction in nucleoli and ribosomal DNA. The reduction in nucleoli was attributable to the inability of the damaged͞ destroyed nucleolar genes to initiate ribosomal RNA synthesis and subsequently produce a nucleolus at the completion of mitosis (6,7). However, in these early studies, the single photon thermal and multiphoton microplasma-induced shock wave damage extended outside the laser focal point, producing secondary damage that reduced cell survival.…”

Laser Microsurgery in Cell and Developmental Biology

“…Three different laser microbeam systems were used in these studies: the low-power argon laser, with acridine orange sensitization; a high-power argon laser, without dye photosensitization (most likely a multiphoton process mechanism); and the fourth harmonic (265 nm) of a neodymium-¥ AG laser. Not only can the nucleolar genes be selectively deleted, causing a loss of nucleoli in the subsequent cell genera-lions, but a corresponding lack of one light-staining Giemsa band in the nucleolar organizer region of the chromosome can be demonstrated in cells cloned from the single irradiated cell (13) (Fig. 4).…”

Technique of laser chromosome welding for chromosome repair and artificial chromosome creation

“…The optical tweezers can also perform chromosome stretching and suspension. There were several studies about using laser micro beams to cut and move chromosome or chromosome fragments [14][15][16][17][18][19][20][21][22][23], and to alter selectively gene expression by removal of an active genetic region [16] or induce DNA interstrand crosslinks, ablate chromosome telomeres and disturb the mitosis process [21]. However, the previously implemented techniques were lacking in detailed information about the correlation between the parameters of micro beam laser and the incision width and quality upon the cut chromosomes.…”