Cellular neuroscienceA tyrosine hydroxylase–yellow fluorescent protein knock-in reporter system labeling dopaminergic neurons reveals potential regulatory role for the first intron of the rodent tyrosine hydroxylase gene
Section snippets
TH targeting vector
A targeting vector was designed to replace exon 1, intron 1, and the first 15 bp of exon 2 of the mouse TH gene with the gene for YFP and a phosphoglycerate kinase-neo gene cassette (Neo) flanked by loxP sites for positive selection through antibiotic resistance. The vector was designed to place the YFP gene in frame with the endogenous ATG start codon at +6. The targeting vector was composed of 5.2 kb of 5′ homology sequence and 0.8 kb of 3′ homology sequence, encompassing the remaining
Generation of TH–YFP mES cell line
In order to generate a reporter system to label dopaminergic neurons we used a knock-in approach to insert cDNA encoding YFP into the TH locus on one allele through homologous recombination in mES cells, thus placing YFP under the direct control of the entire endogenous 5′ upstream region of the mouse TH gene. Because these cells contain one wild-type allele expressing TH and one knock-in allele expressing YFP, we predicted that in this system YFP and TH would be expressed in a nearly identical
Discussion
The initial goal of this work was to generate a reporter system that would accurately label dopamine-expressing neurons and could be used for both in vitro and in vivo studies of dopaminergic differentiation and maintenance as well as for transplantation studies in mouse models of PD. Previous reporter systems using transgenic approaches to express reporter genes from the 5′ flanking sequences of the TH gene have not replicated the endogenous expression pattern of TH with complete accuracy (
Acknowledgments
This research was supported by an Academic Research Initiation grant from the North Carolina Biotechnology Center and a research grant from the Whitehall Foundation (G.F.), Udall Parkinson’s Disease Center of Excellence grant P50 NS39793 (O.I., K.S.K.), National Institutes of Health grant NS22675 (D.M.C.), the Duke University Medical Center Medical Scientist Training Program (B.B.K.) and a fellowship from the Swedish Brain Foundation (E.H.). We thank Dr. Rashmi Chandra, Marybeth Groelle and
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Cited by (13)
Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons
2019, Stem Cell ResearchCitation Excerpt :Furthermore, it demonstrates that the created knock-in faithfully reports TH expression in vitro. This is notable, as previous approaches to a TH-reporter system failed to deliver faithful co-presence of both proteins (Kelly et al., 2006) or were not based on endogenous gene expression (Hedlund et al., 2007). A study was published in 2017 (Xia et al., 2017) with a similar construct for DA neurons to that reported here, using a red fluorescent protein, demonstrating the feasibility of using this kind of reporter system.
A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations
2017, Cell ReportsCitation Excerpt :Developing a method to track TH-positive neurons may enhance identification, sorting, and characterization of iDA/mDA neurons and reduce complexity caused by cell heterogeneity. Previous studies have reported the use of a TH promoter to drive expression of a reporter gene from the predicted TH promoter or by placing a reporter within the exon/intron region of TH in cell lines or mouse strains; in some cases, these transgenes have not yet been fully characterized, and in other cases, they may not completely reproduce endogenous TH expression patterns (Banerjee et al., 1992; Sasaoka et al., 1992; Min et al., 1994; Gelman et al., 2003; Kelly et al., 2006; Gong et al., 2007; Hedlund et al., 2007; Dodd et al., 2011; White and Thomas, 2012; Cui et al., 2016). Here, we describe the generation of a TH reporter that faithfully mimics endogenous TH expression in cell culture and in a rat transplantation model.
Cooperation of nuclear fibroblast growth factor receptor 1 and Nurr1 offers new interactive mechanism in postmitotic development of mesencephalic dopaminergic neurons
2012, Journal of Biological ChemistryCitation Excerpt :One NBRE-like site upstream of the TATA-box (Fig. 6A) was shown to bind FGFR1 (61). Previous reports also suggest that first intron of the TH gene is critical for differential gene regulation among species (48) and can bind Nurr1 (49). In our study, we analyzed Nurr1 and FGFR1 binding to NBRE-like sequence in the TH gene intron-1 (NBS 1; Fig. 6A), as well as the TH promoter region, which contains a sequence homologous to one core element of NurRE (NBS 2; Fig. 6A).
Epigenetic, transcriptional and posttranscriptional regulation of the tyrosine hydroxylase gene
2011, International Journal of Developmental NeuroscienceCitation Excerpt :Substitution of this area with the reporter gene in one of the TH loci resulted in a disturbed expression pattern of the recombined gene in transgenic mice (Kelly et al., 2006). The high acetylation level of histone lysine residues and the presence of sites hypersensitive to DNase I implies an open chromatin structure in this area and the presence of binding sites for transcriptional factors (Kelly et al., 2006; Romano et al., 2007). TH regulation at the level of transcription elongation is mediated by three RNA polymerase II arrest sites.