GT是指在基因组的原位实现对基因的定点突变

1、1会计学GT是指在基因组的原位实现对基因的定是指在基因组的原位实现对基因的定点突变点突变Two approaches for GT：1. Homologous recombination-dependent gene targeting, e.g. tobacco(ALS乙酰乳酸合成酶 ) , Arbidopsis(PPO，protoporphyrinogen oxidase原卟啉原氧化酶 ) and rice(Waxy); 2. Chimeric RNA/DNA oligonucleotide-directed targeted point mutations, e.g. AHAS(乙酰羟酸

2、合成酶) in maize and ALS in tobacco and rice. Copyright 2007 American Society of Plant BiologistsTerada, R., et al. Plant Physiol. 2007;144:846-856Integration events of a transgene associated with homology-dependent GT with positive-negative selection Copyright 2007 American Society of Plant Biologists

3、Terada, R., et al. Plant Physiol. 2007;144:846-856Strategy for the modification of the Adh locusZinc-finger nucleases. (a) Zinc fingers are depicted that recognize nucleotide triplets of a target sequence. Multiple fingers can be joined together to create zinc-finger proteins that recognize extended

4、 sequence patterns. (b) Zinc-finger domains are fused to a type II restriction endonuclease such as FokI. (c) When the FokI monomers are brought into proximity by DNA binding, a functional nuclease is created that cleaves the target sequence.ZFN homodimer binding to DNA. Shown is a three-finger zinc

5、 finger linked to the Fn domain through a flexible peptide linker.At the N-terminus of each ZFN resides a nuclear localization signal (NLS). The Fn domain is linked to the C-terminal finger (in this case finger 3) of the zinc finger domain. For most efficient cleavage there is no amino acid linker b

6、etween the zinc finger domain and the Fn domain. The binding sites are arranged in an inverted orientation so that one ZFN is making most of its major contacts with one strand of DNA, whereas the other ZFN is making most of its major contacts with the other strand of DNA. Between the two binding sit

7、es is a nucleotide spacer (NNN.), the sequence of which does not seem to be important. This figure is a modification of Figure 2c from Jantz et al.47.Nature Biotechnology 23, 967 - 973 (2005) Molecular reagents for measuring homologous recombination. (a) pDW1273 encodes a functional GUS:NPTII report

8、er gene. AI denotes the artificial intron within the GUS coding sequence. The ColE1 replicon and Ampr gene are for recovery of the integrated construct by plasmid rescue. The GUS:NPTII coding sequence in pDW1363 has a 600 bp deletion that includes GUS and NPTII coding sequences critical for function

9、 (mutant forms of GUS and/or NPTII are indicated by lower-case letters). A Zif268 recognition site (depicted in Figure 1) is inserted at the site of the deletion. The Hygr marker functions in plants and can be used to select cells carrying the reporter construct. Filled triangles depict the left and

10、 right borders of the T-DNA. Open arrows indicate the primers used for the PCR reactions in Figure 4. The donor DNA, pDW1269, lacks sequences 5 of the artificial intron and is used to repair the GUS:NPTII reporter in pDW1363 by homologous recombination. The donor DNA has a diagnostic XhoI restrictio

11、n site. (b) The target gene and donor DNA are shown undergoing recombination. Numbers adjacent to the open arrows indicate the size of expected PCR products. The length of homology between donor and target is given below the donor DNA.Outline of zinc finger nuclease (ZFN) functional assays and their

12、 vector systems.The step-by-step comprehensive analysis of novel ZFNs is composed of four distinct assays (left panel), each based on monitoring ZFN activity using a defined set of vectors (right panel). The in vitro digestion assay tests the digestion activity of an Escherichia coli-expressed ZFN o

13、n its recognition site cloned on a target vector. The T-DNA repair assay requires the assembly of a dual-expression cassette on an Agrobacterium tumefaciens binary vector and tests the ability of a constitutively expressed ZFN to digest and repair a mutated GUS reporter gene cloned on the T-DNA regi

14、on of that vector. The transgene repair assay calls for separating the ZFN expression cassette from its target site based on the activation of a mutated GUS reporter gene in transgenic calli, while the whole-plant repair assay activates the mutated GUS reporter gene in seedling or mature plant tissu

15、es upon specific activation of the ZFN. TS, ZFN target site; KAN, kanamycin; hsp, heat shock promoter. Copyright 2007 American Society of Plant BiologistsDafny-Yelin, M., et al. Plant Physiol. 2007;145:1118-1128Methods for the assembly of multiple-gene binary plasmids Copyright 2007 American Society

16、 of Plant BiologistsDafny-Yelin, M., et al. Plant Physiol. 2007;145:1118-1128The general structure of a pSAT-based plant expression vectorTwo approaches for GT：1. Homologous recombination-dependent gene targeting, e.g. tobacco(ALS乙酰乳酸合成酶 ) , Arbidopsis(PPO，protoporphyrinogen oxidase原卟啉原氧化酶 ) and ric

17、e(Waxy); 2. Chimeric RNA/DNA oligonucleotide-directed targeted point mutations, e.g. AHAS(乙酰羟酸合成酶) in maize and ALS in tobacco and rice.Zinc-finger nucleases. (a) Zinc fingers are depicted that recognize nucleotide triplets of a target sequence. Multiple fingers can be joined together to create zinc

18、-finger proteins that recognize extended sequence patterns. (b) Zinc-finger domains are fused to a type II restriction endonuclease such as FokI. (c) When the FokI monomers are brought into proximity by DNA binding, a functional nuclease is created that cleaves the target sequence.ZFN homodimer binding to DNA. Shown is a three-finger zinc finger linked to the Fn domain through a flexible peptide linker.At the N-terminus of each ZFN resides a nuclear localization signal (NLS). The Fn domain is linked to