Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.

We report gene targeting via homologous recombination in zebrafish. We co-injected fertilized eggs with transcription activator-like effector nuclease mRNAs and a donor vector with long homologous arms targeting the tyrosine hydroxylase (th) locus, and we observed effective gene modification that was transmitted through the germ line. We also successfully targeted two additional genes. Homologous recombination in zebrafish with a dsDNA donor expands the utility of this model organism.

ABSTRACT

Complementary and genomic DNA for the murine transferrin receptor 2 (TfR2) were cloned and mapped to chromosome 5. Northern blot analysis showed that high levels of expression of murine TfR2 occurred in the liver, whereas expression of TfR1 in the liver was relatively low. During liver development, TfR2 was up-regulated and TfR1 was down-regulated. During erythrocytic differentiation of murine erythroleukemia (MEL) cells induced by dimethylsulfoxide, expression of TfR1 increased, whereas TfR2 decreased. In MEL cells, expression of TfR1 was induced by desferrioxamine, an iron chelator, and it was reduced by ferric nitrate. In contrast, levels of TfR2 were not affected by the cellular iron status. Reporter assay showed that GATA-1, an erythroid-specific transcription factor essential for erythrocytic differentiation at relatively early stages, enhanced TfR2 promoter activity. Interestingly, FOG-1, a cofactor of GATA-1 required for erythrocyte maturation, repressed the enhancement of the activity by GATA-1. Also, CCAAT-enhancer binding protein, which is abundant in liver, enhanced the promoter activity. Thus, tissue distribution of TfR2 was consistent with the reporter assays. Expression profiles of TfR2 were different from those of TfR1, suggesting unique functions for TfR2, which may be involved in iron metabolism, hepatocyte function, and erythrocytic differentiation. IntroductionIron, one of the essential elements of life, is carried by transferrin (Tf) in the serum and absorbed by the cells through transferrin receptor (TfR1)-mediated mechanisms. 1 Diferric Tf in the serum binds to TfR1 on the cell surface, followed by endocytosis of the receptor-ligand complex. HFE is an atypical major histocompatibility complex (MHC) class I molecule that can form a complex with TfR1 on the cell surface and interfere with binding of Tf to TfR1. [2][3][4] Mutations of HFE are thought to be related to hereditary hemochromatosis. After internalization of the Tf-TfR1 complex, iron is released from the Tf-TfR1 complex in the endosome and is transported to the cytoplasm and mitochondria by DMT1/Nramp2, a transmembrane iron transporter. [5][6][7][8] Recently, we cloned human TfR2, another transferrin receptor gene. Two alternatively spliced forms of human TfR2 transcripts were identified: ␣ and ␤. TfR2-␣ was highly expressed in K562, an erythroid leukemia cell line; in liver; and in HepG2, a hepatoma cell line. 9 Increased Tf binding and iron uptake were observed in Chinese hamster ovary cells that had been stably transfected with human TfR2-␣. Although human TfR2-␣ and TfR1 are very similar in their primary structure and Tf-binding property, the expression pattern of TfR2 mRNA is quite different from that of TfR1. We have found that in K562 cells, iron chelation up-regulates TfR1 and down-regulates TfR2, and iron overload down-regulates TfR1 and slightly up-regulates TfR2. 10 TfR1 knockout mice did not survive beyond embryonic day 12.5 because of severe anemia and neurologic abnormalities, which indicates that murine TfR2 cannot ...