Homologous recombination in plant cells after Agrobacterium-mediated transformation.

Abstract

A single amino-acid change in the acetolactate synthase (ALS) protein of tobacco confers resistance to the herbicide chlorsulfuron. A deleted, nonfunctional fragment from the acetolactate synthase gene, carrying the mutant site specifying chlorsulfuron resistance plus a closely linked novel restriction site marker, was cloned into a binary vector. Tobacco protoplasts transformed with Agrobacterium tumefaciens carrying this vector yielded chlorsulfuron-resistant colonies. DNA gel blot analysis of DNA from these colonies suggested that in three transformants homologous recombination had occurred between the endogenous ALS gene and the deleted ALS gene present in the incoming T-DNA. Plants were regenerated from these chlorsulfuron-resistant colonies, and in two of the transformants, genetic analysis of their progeny showed that the novel gene segregated as a single Mendelian locus. Possible models for the generation of these recombinant plants are discussed.

Site‐specific integration of DNA into wild‐type and mutant lox sites placed in the plant genome

“…Gene integration via homologous recombination, though successful in fungal and animal systems, is not yet practical in plants. DNA integration into the plant genome through homologous recombination occurs at very low frequencies compared with the high background of illegitimate recombination events (Halfter et al, 1992;Lee et al, 1990;Miao and Lam, 1995;Offringa et al, 1990;Paszkowski et al, 1988). …”

A NEWMOSSGENETICS: Targeted Mutagenesis inPhyscomitrella patens

“…In 1989, the observation that the ratio of targeted to random integration events upon transformation of a mouse embryonic stem (ES) cell line ranges from 0.1-10% (16,123) accounts for its exponential development as a model system in animal biology (77). Since 1988, the feasibility of gene targeting in plants has been demonstrated, following either direct gene transfer to protoplasts (87) or Agrobacterium-mediated transformation (65,80) into either artificial (80,87) or natural loci (65). Yet, despite numerous studies, the ratio of targeted to random integration events observed so far in plants hardly reaches 10 −4 , which prevents the general use of gene targeting approaches for plant functional genomics (74,89,126).…”

An efficient gene targeting system using homologous recombination in plants

“…식물에서는 담배에서 처음으로GT을 성공하였으 며 (Paszkowski et al 1988), 다른 다양한 식물에서도HR을 이용한GT에 대한 연구가 활발하게 수행되고 있다 (Terada et al 2007;Puchta and Fauser 2013;Voytas 2013;Endo and Toki 2014). 하지만 식물에서는 HR을 이용한GT가 이루어 지는 빈도가 동물에 비해 매우 낮기 때문에 보편적으로 이용되기가 어려웠다 (Lee et al 1990;Terada et al 2002;Hohn and Puchta 2003;Endo et al 2006b;Endo et al 2007). Positive-negative selection system을 이용한GT Positive-negative selection system을 이용한GT은 쥐의 knockout 돌연변이를 효율적으로 만드는 방법으로 개발되었다 (Mansour et al 1988).…”

“…그러나 손상된 DNA부위를 간단히 접합하는 방식으로 복구하는 NHEJ 경로 (Fig. 1A)로 대부분 복구되어 HR을 통한 DNA 복구 빈도가 매우 낮기 때문에 HR을 이용한 GT의 성공 율은 형질전환체의 10 -3 에서 10 -6 에 그치고 있다 (Lee et al 1990;Terada et al 2002;Hohn and Puchta 2003;Endo et al 2007). GT의 효율을 높이는 방법으로 인위적으로 DNA 손상 을 유도하고 HR의 빈도를 높이는 전략이 시도되었다.…”