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A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish.

Quach HN, Tao S, Vrljicak P, Joshi A, Ruan H, Sukumaran R, Varshney GK, LaFave MC, Ds Screen TeamBurgess SM, Winkler C, Emelyanov A, Parinov S, Sampath K - G3 (Bethesda) (2015)

Bottom Line: However, understanding the functions of all the genes in the genome remains a challenge.Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes.We obtained 642 zebrafish lines with dynamic reporter gene expression.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604.

No MeSH data available.


Related in: MedlinePlus

Mapping of DsDELGT4 integrations to zebrafish chromosomes. Two hundred seventy-seven integrations sites (identified by TAIL-PCR from 283 founder lines) were used for this analysis. (A) Relative positions of integration sites on each chromosome according to zebrafish reference genome Zv9. (B) Distribution of insertions in specific regions of annotated genes (n = 277 insertions). (C–F) Alignment of 28 nucleotides around the integration sites located in intronic (C; n = 131), exonic (D; n = 30), or intergenic regions (E; n = 116) compared to all mapped Ds insertion sites (F). The eight nucleotides duplicated at integration sites are at nucleotide positions 11 to 18.
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fig5: Mapping of DsDELGT4 integrations to zebrafish chromosomes. Two hundred seventy-seven integrations sites (identified by TAIL-PCR from 283 founder lines) were used for this analysis. (A) Relative positions of integration sites on each chromosome according to zebrafish reference genome Zv9. (B) Distribution of insertions in specific regions of annotated genes (n = 277 insertions). (C–F) Alignment of 28 nucleotides around the integration sites located in intronic (C; n = 131), exonic (D; n = 30), or intergenic regions (E; n = 116) compared to all mapped Ds insertion sites (F). The eight nucleotides duplicated at integration sites are at nucleotide positions 11 to 18.

Mentions: Dynamic reporter expression is usually associated with protein functions of tagged genes, so it is important to identify the corresponding insertions. However, most of the reporter lines have complex protein trap and/or enhancer trap expression patterns due to the tagging of many genes by multiple Ds insertions, making it difficult to assign each specific expression pattern to a particular insertion by a single method. We used TAIL-PCR to recover the flanking sequences of the Ds insertions, performed 5′ rapid amplification of cDNA ends (5′ RACE) to identify the trapped genes, and we also utilized a high-throughput sequencing method in cases where both TAIL-PCR and RACE failed. We mapped 467 sequences from 310 reporter-positive lines by TAIL-PCR and RACE and found them to be distributed across all 25 chromosomes with no obvious bias or recognizable hotspots (based on Zv9; Figure 5A). We identified 277 unique Ds insertion sites. Fish with 212 of the insertions were successfully propagated and sperm has been cryopreserved. Approximately 42% of the 277 insertions map to intergenic regions, but the majority were found in introns (47%), exons (7%), or UTRs of known genes (4%), some of which potentially lead to protein trap events (Figure 5B).


A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish.

Quach HN, Tao S, Vrljicak P, Joshi A, Ruan H, Sukumaran R, Varshney GK, LaFave MC, Ds Screen TeamBurgess SM, Winkler C, Emelyanov A, Parinov S, Sampath K - G3 (Bethesda) (2015)

Mapping of DsDELGT4 integrations to zebrafish chromosomes. Two hundred seventy-seven integrations sites (identified by TAIL-PCR from 283 founder lines) were used for this analysis. (A) Relative positions of integration sites on each chromosome according to zebrafish reference genome Zv9. (B) Distribution of insertions in specific regions of annotated genes (n = 277 insertions). (C–F) Alignment of 28 nucleotides around the integration sites located in intronic (C; n = 131), exonic (D; n = 30), or intergenic regions (E; n = 116) compared to all mapped Ds insertion sites (F). The eight nucleotides duplicated at integration sites are at nucleotide positions 11 to 18.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478556&req=5

fig5: Mapping of DsDELGT4 integrations to zebrafish chromosomes. Two hundred seventy-seven integrations sites (identified by TAIL-PCR from 283 founder lines) were used for this analysis. (A) Relative positions of integration sites on each chromosome according to zebrafish reference genome Zv9. (B) Distribution of insertions in specific regions of annotated genes (n = 277 insertions). (C–F) Alignment of 28 nucleotides around the integration sites located in intronic (C; n = 131), exonic (D; n = 30), or intergenic regions (E; n = 116) compared to all mapped Ds insertion sites (F). The eight nucleotides duplicated at integration sites are at nucleotide positions 11 to 18.
Mentions: Dynamic reporter expression is usually associated with protein functions of tagged genes, so it is important to identify the corresponding insertions. However, most of the reporter lines have complex protein trap and/or enhancer trap expression patterns due to the tagging of many genes by multiple Ds insertions, making it difficult to assign each specific expression pattern to a particular insertion by a single method. We used TAIL-PCR to recover the flanking sequences of the Ds insertions, performed 5′ rapid amplification of cDNA ends (5′ RACE) to identify the trapped genes, and we also utilized a high-throughput sequencing method in cases where both TAIL-PCR and RACE failed. We mapped 467 sequences from 310 reporter-positive lines by TAIL-PCR and RACE and found them to be distributed across all 25 chromosomes with no obvious bias or recognizable hotspots (based on Zv9; Figure 5A). We identified 277 unique Ds insertion sites. Fish with 212 of the insertions were successfully propagated and sperm has been cryopreserved. Approximately 42% of the 277 insertions map to intergenic regions, but the majority were found in introns (47%), exons (7%), or UTRs of known genes (4%), some of which potentially lead to protein trap events (Figure 5B).

Bottom Line: However, understanding the functions of all the genes in the genome remains a challenge.Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes.We obtained 642 zebrafish lines with dynamic reporter gene expression.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604.

No MeSH data available.


Related in: MedlinePlus