Physiological roles of CIDEs CIDEA deficient mice exhibit lean phenotype and are obesity resistant 4

Chapter Chapter Generation of Cidea and Cideb null mice 58 Chapter 3.1 Introductions to gene targeting 3.1.1 History Genetically modified animals including transgenic and knockout models are very powerful tools for gene function research. The first publications of such techniques appeared in 1980 (transgenic) and 1991 (knockout) respectively (Gordon and Ruddle, 1982; Palmiter et al., 1982a; Palmiter et al., 1982b; Petri, 1982; Roemer et al., 1991). The introduction of the mouse embryonic stem cells in the late 1980s (Evans and Kaufman, 1981) and the creation and successful use of vectors for homologous recombination in the early 1990s have made it practical to create mice either by site directed mutations or by exon deletions (knockout) of a specific gene. 3.1.2 Embryonic stem cells Embryonic stem (ES) cells are isolated from inner cell mass of mouse blastocysts (~ day mice embryos). These ES cells can be propagated and cultured on a layer of feeder fibroblast cells and growth media containing leukemia inhibitory factor (LIF), which prevents differentiation and thus ensuring the maintenance of pluripotency. ES cell culture is a critical step of successful creation of a knockout animal. The first established mouse ES cell lines were made from the 129 strain (Evans and Kaufman, 1981), and most knockout mice are created from ES cells of this strain. The coat color of 129 strains are agouti (light brown), and the use of blastocysts from C57BL/6J mice, the coat color of which is black, makes the identification of chimeras easy. Chimeras generated are further tested for germline transmission by cross breeding with C57BL/6J mice. Agouti coat color mice from the crosses are heterozygous for 129/C57BL, which means half their genome was derived from the ES 59 Chapter cells. The germline transmission chimeras can be crossbred with 129 to obtain pure 129 inbred genetic background mice. Alternatively the heterozygous of 129/C57BL mice can be crossbred with C57BL/6J mice for several generations to get pure C57BL/6J inbred mice. 3.1.3 Gene targeting vectors Gene targeting is achieved by the introduction of exogenous added targeting construct into a genomic locus by homologous recombination. Exogenous DNA introduced as such may be randomly integrated into the genome, inserted by homologous recombination or degraded. The efficiency of homologous recombination is primarily dependent on the length of the homologous region between the construct and the genomic locus (Hasty et al., 1991). The homologous regions used should be long enough to ensure efficient recombination, but short enough to be cloned and maintained in a bacterial vector, with reliable options for PCR and Southern blot analysis for the screening of clones. A PCR analysis is rapid and can save time during the primary screen. Southern blot analysis relies on a well-defined restriction enzyme digestion plan and a correspondingly specific probe. Eventually, positive clones from a PCR screen has to be further analysed by Southern blot analysis to eliminate randomly integrated clones. A well-planned Southern blotting strategy with unambiguous outcomes has to be envisioned in detail prior to any gene targeting experiments. 60 Chapter Figure 10 schematic diagrams of simple gene targeting vector and screening strategy.I, II, III stands for the exons, arrow stands for the primers used for PCR method screening. Homologous recombination can be made with either an insertion vector or a replacement vector. Insertion vector targeting results in duplication of the homologous region and an insertion of non-homologous sequences, whereas replacement vector targeting results in the insertion of non-homologous sequences in the middle of the homologous region. Replacement vectors are by far the most commonly used, and are the types used in this project. In a replacement vector, a negative selection marker placed outside the homologous region will not be integrated by a correct homologous recombination. This makes it possible to eliminate cells with random targeting construct integration. Two types of negative selection markers are commonly used, the Xanthine/guanine phosphoribosyl transferase gene (gpt) and the herpes simplex virus thymidine kinase gene (HSVtk). Both genes encode enzymes that convert a non-toxic drug into a toxic derivative. Cells expressing the gene for negative selection are therefore killed by the derivative whereas cells that not would survive. The drug gancyclovir is used for HSVtk selection while 6-thioxantine or 6-thioguanine is used for gpt selection. When both the positive and negative selection drugs are added to the 61 Chapter media after transfection, the cell population will be enriched in cells where the replacement construct has been integrated into the genome via homologous recombination. In a typical conventional replacement vector, the coding region (or part of it) in the middle of the homologous region is replaced by an antibiotic resistance gene cassette (containing a promoter sequence and a transcriptional termination region). This ensures that any resulting mRNA from the original locus will not encode a functional protein. The antibiotic resistance marker gene is used in the selection for positive clones containing a homologous recombined allele of the gene of interest. The neomycin (neo) resistance gene is a commonly used marker for antibiotic selection of mammalian cells. The neo resistance gene confers resistance to neomycin or geniticin (G418). 3.2 Mouse Cideb genomic mapping and cloning After screening a mouse 129/Sv genomic library using mouse full length Cideb cDNA as a probe, seven positive phage clones were obtained. Denatured phage DNA samples from all these clones were spotted onto Hybond-N nylon membrane (Amersham) and were probed with γ32p ATP end labeled oligonucleotide primers m453, P4 (Cideb coding region nt 1-24), P2 (Cideb coding region nt 313-337) and P6 (Cideb coding region nt 610-634). All seven clones were P2 and P6 positive (data not shown), and therefore all clones contain at least a part of the Cideb cDNA sequence. Their restriction enzyme digestion pattern also showed that these clones were similar in size. One of the clones was chosen for further analysis of the Cideb genomic structure. A DNA insert of about 18 kb was released from the phage clone by Not I digestion and subcloned into pBluescript, and analysed in detail by subcloning, restriction enzyme digestion, Southern blotting and DNA sequencing (as described in 62 Chapter Chapter 2). Part of the restriction enzyme map and genomic structure of Cideb is shown in Figure 11 (top panel). 1kb Wild- type allele AvrII 5’ ATG probe AvrII NcoI PstI TAA AvrII BamHI 3’ HindIII Targeting vector TK Neo Targeted allele AvrII 3’ 5’ AvrII Neo Figure 11 Targeted Disruption of the Cideb Gene. Schematic representation of the Cideb gene targeting strategy. Top: partial linear restriction map of the Cideb locus. Middle: The Cideb gene-targeting construct. Bottom: The expected mutant locus. The corresponding DNA fragment used as a probe for Southern blot analysis is also shown below the top as well as in the bottom linear maps. Recently, the mouse genome sequence has been made available online. The cDNA sequence of Cideb was therefore used in BLAST search alignments to obtain the corresponding genomic sequence. Part of the BLAST result is shown in Figure 12A and Figure 12B. The mouse genomic Cideb locus is found in Mus musculus chromosome 14. The genomic DNA structure of Cideb was therefore confirmed to have been correctly mapped in our own attempts. 63 Chapter Figure 12A Genomic DNA sequence of the 5’ region of Cideb gene. The first two exons were contained in this region. This part of the DNA fragment was not contained in the 18 kb fragment obtained in our screening. 64 Chapter Figure 12B Genomic DNA sequence of the 3’ region of Cideb gene. This gene fragment containing the C-terminus exons of Cideb was subcloned, mapped and sequenced. This part of the gene was replaced by neomycin resistant marker gene in the Cideb replacement construct as shown in figure 11. 65 Chapter 3.3 Generation of Cideb null mice The gene targeting strategy for Cideb knockout is shown in Figure 11. Cideb deficient mice were generated by having the three C-terminal exons replaced by the neomycin resistant gene, which also served as a positive selection marker. A 1.6 kb fragment (NcoI-PstI) at the 5’ end of the genomic locus was used as the short arm and a 7.2kb fragment (BamHI-HindIII) of the 3’ end of the genomic locus was used as the long arm (shown in Figure 11, middle panel). Correct targeting via homologous recombination was verified by Southern blot analysis using AvrII digestion of the genomic DNA and probed by the AvrII-NcoI DNA fragment shown in Figure 11 (top panel). 250 geneticin resistant clones were isolated and screened and 17 positive clones were identified. Three of the targeted ES cell clones were selected for injection into C57BL/6J mouse blastocysts to generate chimeric mice. Because the ES cells are derived from male mice, 21 viable chimeras were generated and all were male. The chimeric mice were subsequently crossbred with wild type C57BL/6J mice to obtain germline transmission of the targeted allele. Cideb heterozygous mice were fertile and were crossbred to obtain mice homozygous for Cideb deletion. The mice genotypes were confirmed by Southern blot analysis as shown in Figure 13A. Accordingly, the wild type (Cideb +/+ ) mice should show a band 3.2 kb in size whereas Cideb-/- mice should exhibit a 5.0 kb size fragment after digestion by AvrII and probed with the DNA fragment shown in Figure 11. Cideb+/+, Cideb+/- and Cideb-/- mice were born at an expected Mendelian frequencies and no apparent growth abnormality was observed. To confirm functional deletion of Cideb, Western blot analysis was performed. As shown in Figure 13B, no 66 Chapter Cideb protein was detected in the Cideb-/- mice. Thus, the Cideb locus has been successfully disrupted. Phenotypic analysis of Cideb null mice is not the focus of this thesis and will not be described further. A +/+ +/- -/5.0kb 3.2kb B +/+ -/Anti-Cideb Anti-Tubulin Figure 13 Inactivation of Cideb in mouse. (A) The correct targeting of the mouse Cideb locus was verified by Southern blot analysis of AvrII digested mice tail DNA. The +/+ genomic DNA has a 3.2 kb labeled fragment while the -/- genomicDNA has a 5.0 kb labeled fragment. (B) The inactivation of Cideb gene was further demonstrated by western blot analysis using anti-Cideb antibody (upper panel). 100mg of protein lysate from mouse liver were loaded in each lane. Anti-tublin was used for protein loading normalization (lower panel). 3.4 Mouse Cidea genomic map and gene targeting strategy To elucidate the biological role of Cidea in vivo, mouse Cidea genomic DNA was isolated after screening a mouse 129/Sv genomic library using mouse full length Cidea cDNA as a probe. Mouse Cidea gene is located at Mus musculus chromosome 18. The gene spans a region of 10 kb and consists of six exons in total. Part of the genomic restriction enzyme map and structure was shown in Figure 14 (top panel). As shown in Figure 14 (middle panel), the Cidea targeting construct was designed to remove regions encompassing part of the first intron and the second exon. Deletion of this region will result in the disruption of the reading frame and the 67 Chapter generation of a severely truncated Cidea protein containing only the first eight amino acid residues. 1kb 5’ Wild- type allele EcoRI ATG PstI EcoRI NcoI 3’ EcoRI NcoI probe TAA Targeting vector TK Neo 3’ Targeted allele EcoRI 5’ EcoRI EcoRI Neo NcoI NcoI NcoI Figure 14 Targeted Disruption of the Cidea Gene. Schematic representation of the gene targeting strategy. Top: partial restriction map of the Cidea locus. Middle: construct of Cidea gene targeting vector. Bottom: the expected mutant locus. DNA fragment used as a probe for Southern blotting is also indicated under the top figure. Three positive clones were identified among 250 geneticin resistant ES cell clones isolated and screened. All the three targeted ES cell lines were injected into C57BL/6J mouse blastocysts to generate chimeric mice. 19 viable chimeras were generated and 17 were male. All 17 males were mated with female C57BL/6J mice and the offsprings were tested for germline transmission of the homologous recombined allele. All 17 males turned out to be germline-transmitted chimeras. The Cidea heterozygous mice derived were phenotypically normal. These were then crossbred to obtain mice homozygous for Cidea deletion. 68 Chapter Southern Northern +/- +/+ -/5.0 kb 2.6 kb +/+ -/- Cidea β-Actin +/+ Western -/Cidea UCP1 Figure 15 Deletion of Cidea in mice. Southern: The correct targeting of the mouse Cidea locus was verified by Southern blot analysis of NcoI digested mice tail DNA. The +/+ genomic DNA has a 5.0kb fragment while the -/- genomic DNA has a 2.6 kb fragment. Northern: The inactivation of Cidea gene was further demonstrated by Northern blot analysis using Cidea cDNA as a probe and β-Actin for normalization of RNA loading. Western: The functional inactivation of Cidea gene was further demonstrated by Western blot analysis using anti-Cidea antibody, UCP1 was used as a control. 100mg of protein lysate from mouse BAT were loaded in each lane. As shown in Figure 15, the mice genotypes were confirmed by Southern blot analysis. Accordingly, the predicted Cidea+/+ mice had a 5.0 kb size fragment whereas Cidea-/- mice had a 2.6 kb size fragment after digestion by NcoI and probed with the probe described in Figure 14. Northern blot analysis also revealed no Cidea mRNA transcript in Cidea null mice. The deficiency in Cidea protein in Cidea-/- mice was further confirmed by Western blot analysis (Figure 15, lower panel). The transcription and translation of Cidea gene was thus successfully inactivated in Cidea-/- mice (or Cidea null mice). The first generation of heterozygous mice breeding yielded 14 (+/+), 31(+/-) and 15(-/-) mice, thus showing the expected Mendelian ratios of heterozygous and 69 Chapter homozygous descendents. Cidea-/- mice appear to be normal and fertile. No apparent developmental, anatomical or behavioral defects were observed. 70 [...]... translation of Cidea gene was thus successfully inactivated in Cidea- /- mice (or Cidea null mice) The first generation of heterozygous mice breeding yielded 14 (+/+), 31(+/-) and 15(-/-) mice, thus showing the expected Mendelian ratios of heterozygous and 69 Chapter 3 homozygous descendents Cidea- /- mice appear to be normal and fertile No apparent developmental, anatomical or behavioral defects were observed... kb size fragment whereas Cidea- /- mice had a 2.6 kb size fragment after digestion by NcoI and probed with the probe described in Figure 14 Northern blot analysis also revealed no Cidea mRNA transcript in Cidea null mice The deficiency in Cidea protein in Cidea- /- mice was further confirmed by Western blot analysis (Figure 15, lower panel) The transcription and translation of Cidea gene was thus successfully... +/+ -/- Cidea β-Actin +/+ Western - /Cidea UCP1 Figure 15 Deletion of Cidea in mice Southern: The correct targeting of the mouse Cidea locus was verified by Southern blot analysis of NcoI digested mice tail DNA The +/+ genomic DNA has a 5.0kb fragment while the -/- genomic DNA has a 2.6 kb fragment Northern: The inactivation of Cidea gene was further demonstrated by Northern blot analysis using Cidea. .. viable chimeras were generated and 17 were male All 17 males were mated with female C57BL/6J mice and the offsprings were tested for germline transmission of the homologous recombined allele All 17 males turned out to be germline-transmitted chimeras The Cidea heterozygous mice derived were phenotypically normal These were then crossbred to obtain mice homozygous for Cidea deletion 68 Chapter 3 Southern... as a probe and β-Actin for normalization of RNA loading Western: The functional inactivation of Cidea gene was further demonstrated by Western blot analysis using anti -Cidea antibody, UCP1 was used as a control 100mg of protein lysate from mouse BAT were loaded in each lane As shown in Figure 15, the mice genotypes were confirmed by Southern blot analysis Accordingly, the predicted Cidea+ /+ mice had... generation of a severely truncated Cidea protein containing only the first eight amino acid residues 1kb 5’ Wild- type allele EcoRI ATG PstI EcoRI NcoI 3’ EcoRI NcoI probe TAA Targeting vector TK Neo 3’ Targeted allele EcoRI 5’ EcoRI EcoRI Neo NcoI NcoI NcoI Figure 14 Targeted Disruption of the Cidea Gene Schematic representation of the gene targeting strategy Top: partial restriction map of the Cidea locus... Middle: construct of Cidea gene targeting vector Bottom: the expected mutant locus DNA fragment used as a probe for Southern blotting is also indicated under the top figure Three positive clones were identified among 250 geneticin resistant ES cell clones isolated and screened All the three targeted ES cell lines were injected into C57BL/6J mouse blastocysts to generate chimeric mice 19 viable chimeras . (Evans and Kaufman, 1981), and most knockout mice are created from ES cells of this strain. The coat color of 129 strains are agouti (light brown), and the use of blastocysts from C57BL/6J mice, . inactivated in Cidea -/- mice (or Cidea null mice) . The first generation of heterozygous mice breeding yielded 14 (+/+), 31(+/-) and 15(-/-) mice, thus showing the expected Mendelian ratios of heterozygous. Figure 14 Targeted Disruption of the Cidea Gene. Schematic representation of the gene targeting strategy. Top: partial restriction map of the Cidea locus. Middle: construct of Cidea gene