embryo cdna library  (TaKaRa)

Journal: Scientific reports

Article Title: Analyses of binding partners and functional domains for the developmentally essential protein Hmx3a/HMX3.

doi: 10.1038/s41598-023-27878-9

Figure Lengend Snippet: Figure 1. Isolation of novel binding partners of HMX3 with yeast two-hybrid screening. Top: Schematic representation of bait and prey constructs used in screening. Full-length mouse HMX3 fused to the Gal4 DNA- binding domain (DBD) was used as bait. A library of stage E11 mouse cDNAs fused to the Gal4 activating domain (AD) was used as prey. 7.2 × 108 solid plate matings were performed, yielding 3200 positive colonies. Positive prey inserts were segregated and sequenced and reads were aligned to the mouse genome, identifying 851 unique genes. After removing non-coding genes and those for which only untranslated region (UTR) or intronic sequences were recovered, 539 protein-coding sequences remained. Zebrafish orthologs of the most functionally interesting genes were selected for further analysis with Co-IPs.

Article Snippet: We used mouse E11 cDNA library (630478; Takara Bio) cloned into pGAD57 vector as prey, transformed into Y187 yeast strain.

Techniques: Isolation, Binding Assay, Two Hybrid Screening, Construct

Journal: PLoS ONE

Article Title: At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

doi: 10.1371/journal.pone.0004352

Figure Lengend Snippet: This figure summarizes our results about the imprinting and structure of genes in this mouse region. Arrows denote transcriptional orientations. Genes written in black were shown to be biallelically expressed. Genes drawn in blue are paternally expressed, while those in red are maternally expressed. Genes written in yellow were undetectable by RT-PCR. For Begain transcribed from promoter 2, this transcript was coded blue as it is paternally expressed in sheep in a tissue specific manner, although in this study, RT-PCR failed to amplify a transcript derived from this promoter. Of these, accession numbers, AK141557, AK163826, AK048151, and AK044800, correspond to unspliced “transcripts” whose cDNA sequence ends in a polyA stretch of genomic DNA, suggesting that these “cDNAs” may be genomic DNA contamination in the Riken mouse cDNA libraries. For the bottom panel, exons are dark colored while introns are light. Purple lines represent the position of microRNA precursors. In all, fifty-two mouse microRNA precursors as listed in map to this imprinted region.

Article Snippet: An arrayed mouse day 19 embryo cDNA library (Origene MEA-1001) was screened by PCR to isolate Irm , Meg8 , Meg9 , Peg11 , and anti-Peg11 clones, while an adult mouse brain cDNA library was used for Dlk1 /“DAT” cloning (Origene MAB-1001).

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Sequencing

Journal: PLoS ONE

Article Title: At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

doi: 10.1371/journal.pone.0004352

Figure Lengend Snippet: (A) A DraIII restriction site polymorphism between 129S1 and CzechII/Ei mice was utilized to determine that Dlk1 is paternally expressed. PCR primers were Dlk1 2Up/317Dn. (B) Northern analysis using the same Dlk1 PCR fragment as probe revealed that Dlk1 is widely expressed. Of the tissues investigated, only the adult brain is characterized by having an additional and abundant transcript that is roughly 4.5 kb in length. (C) Several ESTs cluster to the syntenic region of “DAT” ( Dlk1 associated transcript). RT-PCR was done with “DAT” primers 1533Up/2044Dn. Using a EcoO109I restriction site polymorphism, “DAT” was shown to be like Dlk1 in its paternal expression. (D) Northern analysis on adult tissue samples of “DAT” detected a 4.5 kb transcript only in the brain. This band is identical in size to the long transcript of Dlk1 found in the brain. Moreover, the length of this transcript is greater than the distance (∼2.7 kb) between mouse Dlk1 polyA and “DAT” polyA. (E) RT-PCR was performed with various primer sets. Left most panel showed that Dlk1 is expressed in brain, heart and muscle. Middle panel shows that “DAT” is only detectable in brain and is consistent with Northern findings. For the right panel, PCR was performed with a primer set in which the upstream primer ( Dlk1 1237Up) was before the canonical Dlk1 polyA site while the downstream primer was after this site ( Dlk1 1805Dn). A band was detected only in the brain, indicating that “DAT” is an alternatively polyadenylated transcript of Dlk1 . This result was confirmed by cDNA cloning (Accession numbers: EU434914-EU434917).

Article Snippet: An arrayed mouse day 19 embryo cDNA library (Origene MEA-1001) was screened by PCR to isolate Irm , Meg8 , Meg9 , Peg11 , and anti-Peg11 clones, while an adult mouse brain cDNA library was used for Dlk1 /“DAT” cloning (Origene MAB-1001).

Techniques: Northern Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay

Journal: PLoS ONE

Article Title: At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

doi: 10.1371/journal.pone.0004352

Figure Lengend Snippet: (A). A single nucleotide polymorphism in Irm cDNA was identified at 1094A→T between 129S1 and CzechII/Ei mouse strains that creates a NlaIII restriction site in the CzechII/Ei Irm cDNA. RT-PCR was performed with Irm primers 988Up and 1140Dn. Restriction digests of RT-PCR products from intraspecific crosses between these two subspecies by NlaIII demonstrated that Irm is expressed in a monoallelic manner from the maternal allele. (B) An adult tissue (left) and total embryo from different gestational days (right) polyA + RNA blot (left) was hybridized with a Irm/Rian exon 1 cDNA probe. A 2.5 kb transcript was predominantly detected in the brain and to a significantly lesser extent in testis, stomach, and muscle. Higher molecular weight bands of lower relative abundance in comparison to the 2.5 kb message were also seen that reflect the heterogeneity in spliced variants. Since Rian RNA is approximately 5.4 kb, the predominant RNA product from the Irm/Rian promoter is Irm RNA, while Rian accounts for at most 5% of total transcripts. This result is consistent with the relative abundance of ESTs that are specific for each of the alternatively spliced products.

Article Snippet: An arrayed mouse day 19 embryo cDNA library (Origene MEA-1001) was screened by PCR to isolate Irm , Meg8 , Meg9 , Peg11 , and anti-Peg11 clones, while an adult mouse brain cDNA library was used for Dlk1 /“DAT” cloning (Origene MAB-1001).

Techniques: Reverse Transcription Polymerase Chain Reaction, Northern blot, Molecular Weight

Journal: PLoS ONE

Article Title: At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

doi: 10.1371/journal.pone.0004352

Figure Lengend Snippet: Mouse cDNAs Cloned in This Study.

Article Snippet: An arrayed mouse day 19 embryo cDNA library (Origene MEA-1001) was screened by PCR to isolate Irm , Meg8 , Meg9 , Peg11 , and anti-Peg11 clones, while an adult mouse brain cDNA library was used for Dlk1 /“DAT” cloning (Origene MAB-1001).

Techniques: Clone Assay, Northern Blot