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The ZFN-encoding mRNA was injected into one-cell embryos and a high percentage of animals carried the desired mutations and phenotypes. Their research work demonstrated that ZFNs can specifically and efficiently create heritable mutant alleles at loci of interest in the germ line, and ZFN-induced alleles can be propagated in subsequent generations.
In addition, it has been used to engineer stably modified human embryonic stem cell and induced pluripotent stem cell (IPSCs) clones and human erythroid cell lines, [11] [28] to generate knockout C. elegans, [12] knockout rats, [13] knockout mice, [29] and knockout zebrafish. [14] [30] Moreover, the method can be used to generate knockin organisms.
In addition to histidine, a conserved arginine on the second beta strand of the zinc fingers makes contact with the phosphodiester oxygen on the DNA strand. [25] [26] [29] Also serine 75 on the third finger hydrogen bonds to the phosphate between base pairs 7 and 8, as the only backbone contact with the secondary strand of DNA. [25] [26] [29]
Recombination between two DNA sites begins by the recognition and binding of these sites – one site on each of two separate double-stranded DNA molecules, or at least two distant segments of the same molecule – by the recombinase enzyme. This is followed by synapsis, i.e. bringing the sites together to form the synaptic complex.
Zinc fingers were first identified in a study of transcription in the African clawed frog, Xenopus laevis in the laboratory of Aaron Klug.A study of the transcription of a particular RNA sequence revealed that the binding strength of a small transcription factor (transcription factor IIIA; TFIIIA) was due to the presence of zinc-coordinating finger-like structures. [6]
A double-strand break repair model refers to the various models of pathways that cells undertake to repair double strand-breaks (DSB). DSB repair is an important cellular process, as the accumulation of unrepaired DSB could lead to chromosomal rearrangements, tumorigenesis or even cell death. [ 1 ]
Through targeted deletions, the custom ZFN disables the C-C chemokine receptor 5 gene, which encodes a co-receptor that is used by the HIV virus to enter the cell. [60] As a result of the high degree of sequence homology between C-C chemokine receptors this ZFN also cleaves CCR2 , leading to off-target ~15kb deletions and genomic rearrangements.
The DNA is cut 9 nucleotides downstream of the motif on the forward strand, and 13 nucleotides downstream of the motif on the reverse strand, [3] producing two sticky ends with 4-bp overhangs. Its molecular mass is 65.4 kDa, being composed of 587 amino acids.