Lui, H. bacterial artificial chromosome clone was mapped by Southern blot hybridization using murine cDNA fragments. bacterial artificial chromosome fragments were cloned into pBluescript (Stratagene) and fully sequenced. The targeting vector was constructed in pBluescript such that a 600-bp fragment of the genomic locus encoding exon 2 and 3 was replaced by and a neomycin resistance cassette. A herpes simplex virus thymidine kinase cassette was inserted 3 kb upstream of the targeted sequence. E14.1 ES cells were electroporated with the NotI-linearized targeting vector, Rabbit polyclonal to COT.This gene was identified by its oncogenic transforming activity in cells.The encoded protein is a member of the serine/threonine protein kinase family.This kinase can activate both the MAP kinase and JNK kinase pathways. and the transfected cells subjected to G418 and ganciclovir selection. Clones showing homologous recombination were detected by PCR and subsequently confirmed by Southern blot hybridization with the 3 flanking probe indicated in Fig. ?Fig.1a1a after digestion of ES cell DNA with EcoRI. Single integration was verified PROTAC ER Degrader-3 by probing the Southern blot with the neomycin resistance cassette. Correctly targeted ES cell clones were injected into C57BL/6 blastocysts, which were transferred into pseudopregnant foster mothers. Resulting chimeric mice were backcrossed to C57BL/6 mice, and germ line transmission of the targeted allele was confirmed by Southern blot analysis. Open in a separate window Open in a separate window FIG. 1. Generation of mutant mice. (a) Amino acid sequence alignment of murine SLY1, SLY2, and SASH1 proteins is shown. Identical residues are shaded and protein domains are indicated by boxes. The arrows assign the deletion in SLY1d caused by skipping of exons 2 and 3. The asterisk marks phosphorylated Ser27. (b) The organization of part of the murine genomic locus (top), the linearized targeting vector (middle), and the targeted allele (bottom) are shown. The -galactosidase cDNA and the neomycin resistance cassette were inserted between codon 26 (exon 2) and codon 72 (exon 3) of the gene. The -galactosidase cDNA contains a polyadenylation signal. Exon sequences are represented by solid rectangles, intron sequences by a black line (R, EcoRI). (c) Lysates of splenocytes from wild-type (+/+), heterozygous (+/d), and homozygous mutant (d/d) mice were analyzed by Western blot with two independent rabbit polyclonal antisera directed against amino acids 2 to 16 or 131 to 145 PROTAC ER Degrader-3 of the wild-type SLY1 protein. Antisera specific for eIF4 served as loading control. (d) The skipping of exons 2 and 3 was revealed by reverse transcription-PCR with exon 1- and exon 4-specific primers in splenic mRNA from wild-type (+/+) and heterozygous (+/d) as well as homozygous (d/d) mutant mice. (e) Schematic representation of the mutant SLY1d protein. N, nuclear localization signal; blue lines, immunogenic peptides for generation of SLY1-specific antisera. Mice were kept according to national guidelines for animal care in an SPF animal facility. The mice used in these experiments were backcrossed to the C57BL/6 background for 6 to 8 8 generations. Wild-type littermates were used as controls. Genotyping for the mutation was performed by PCR with the following primers: 5-GATCCATGCCAGCGTTACCA-3(WT/forward), 5-TCGCCTTCTATCGCCTTCTTG(Neo/for), and 5-AGTCATAGCTCTCCATCAGC-3(reverse). PROTAC ER Degrader-3 For reverse transcription-PCR analysis of SLY1 mRNA splicing, total RNA from thymus and spleen was extracted using the TRIZOL reagent (Invitrogen Life Technologies), and was reverse-transcribed to cDNA using oligo(dT) primer and SuperScript II (Invitrogen Life Technologies). The following primer set was used for exon skipping analysis: Ex1_for: 5-GAGTAGCAGCCCCAGGC-3, and Ex4_rev: 5-CTGTTGATGTCTGGCGGC-3. Generation of bone marrow chimeric mice. For generation of bone marrow chimeric mice, recipients were lethally irradiated with 6 Gy and intravenously injected with 107 donor bone marrow cells 24 h later. Analysis of mice was performed 4 weeks after adoptive bone marrow transfer. B-cell proliferation. Splenocytes from wild-type and gene by homologous recombination in embryonic stem cells and generated homozygous mutant mice (Fig. ?(Fig.1b).1b). Western blot analyses with two independent antisera raised against the N-terminal (amino acids 2 to 16) and central regions (amino acids 131 to 145) of the SLY1 protein revealed that homozygous mutant mice express a truncated protein of 45 kDa as opposed to the 55-kDa wild-type SLY1 protein (Fig. ?(Fig.1c1c). Based on these findings we examined the possibility that the residual SLY1 PROTAC ER Degrader-3 protein was generated by a splicing event involving exons 1 and 4, thereby skipping the inserted -galactosidase.