Mouse models for inherited endocrine and metabolic disorders

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   Figure 1

   Methods for non-targeted (random) mutagenesis. (A) Chemical mutagenesis using N-ethyl-N-nitrosourea (ENU). ENU is an alkylating agent that transfers its ethyl group to one of a number of reactive sites on DNA nucleotides, including the O⁶ of guanine as shown. Modification of guanine with the ethyl group (red) to produce O⁶-ethylGuanine (O-eG), causes mispairing during DNA replication, e.g. at spermatogenesis, and during subsequent replication a mutation is introduced. (B) ENU-mutagenised G0 male mice, harbouring induced mutations in their sperm DNA, are mated with wild-type females of the same strain to generate G1 mice. G1 males are examined for phenotypic abnormalities (i.e. the phenotype-driven screens). Males with phenotypic abnormalities of interest are then mated with wild-type females to facilitate inheritance testing and genetic mapping in affected offspring (G2) to identify the mutation causing the phenotypic abnormality. DNA and sperm from all the G1 males are also archived to facilitate genotype-driven screens. m (in red), mutant allele; +, wild-type allele. (C) Insertion mutagenesis using gene trap vectors. Gene trap vectors consist of a strong splice acceptor (SA), a reporter gene such as β-galactosidase (β-gal) and a poly-adenine tract (pA). The gene trap randomly inserts into the host genome, and during splicing, the splice acceptor is used in preference to the normal genomic splice sites (splicing pattern shown by red lines). Filled boxes denote coding sequences and open boxes denote non-coding sequences.
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   Figure 2

   Gene targeting by modification of embryonic stem (ES) cells. Totipotent ES cells are isolated from the inner cell mass of a blastocyst (for example from a 129Sv (shown) or C57Bl/6 embryo) and cultured. The targeting vector is transferred to the ES cells, and those in which homologous recombination or integration has been successful are selected. These are injected into the inner cell mass of a blastocyst from a different mouse strain (for example C57BL/6 (shown)), which is transferred to the uterus of a pseudopregnant female. The resulting chimaeric offspring (usually males are selected) are bred with wild type, e.g. C57BL/6 mice (usually females are selected) to achieve germline transmission. m, mutant allele; +, wild-type allele.
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   Figure 3

   Strategies for targeted mutagenesis in embryonic stem (ES) cells. Targeting vectors (blue) typically consist of two ‘arms’ of sequence homologous to the target gene flanking a positive selection cassette such as the neomycin phosphotransferase (Neo^R) gene, and with a negative selection cassette such as the thymidine kinase (TK) gene at one end of the construct. The Neo^R cassette is usually flanked by two LoxP sites (open triangles) so that it may be removed by expression of Cre recombinase after homologous recombination (green lines) with the host (wild type) genome (black). When homologous recombination occurs, the negative selection cassette is lost. Thick lines denote sequences derived from genomic DNA, with filled boxes representing coding exons and open boxes representing non-coding exons; thin lines denote sequences derived from vectors. (A) Conventional knockout. In a typical conventional knockout targeting vector, the Neo^R cassette replaces one or more exons, and is then excised by Cre recombinase after homologous recombination. (B) Conditional or inducible knockout. The Neo^R cassette may be flanked by flippase (FLP) recombinase target (FRT) sites (filled triangles) to allow removal by expression of FLP. Part of the coding region of the gene is also flanked by LoxP sites (open triangles). Thus, when homozygote mutant mice are crossed with mice expressing Cre in a tissue-specific manner, or mice expressing an inducible Cre, the gene is knocked out within the tissue or upon administration of the inducer. (C) Knockin. A specific mutation is introduced into the targeting vector (red asterisk), usually by site-directed mutagenesis. The Neo^R cassette is placed in an intron close to the mutation and excised after homologous recombination, either by introduction of Cre recombinase into the ES cells or by breeding mutant mice with mice expressing Cre ubiquitously.
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   Figure 4

   Role of CDKIs in cell cycle progression. The cell cycle consists of five distinct phases. The first growth or gap (G₁) phase, which entails biosynthesis of key enzymes required for DNA manufacture, precedes the synthesis (S) phase during which DNA is synthesised, which is followed by the second gap (G₂) phase, during which the microtubules required for the mitosis (M) phase are produced. The resting phase (G₀) is a post-mitotic phase during which cells are quiescent and non-proliferative. The G₁ to S phase transition of the cell cycle is tightly regulated by cyclin-dependent kinases (CDKs), their partner cyclins and CDK inhibitors (CDKIs). The E2F family of transcription factors is sequestered by members of the retinoblastoma (RB) gene family. CDK4 and CDK6, in conjunction with cyclin D, phosphorylate RB family members, releasing the E2F family members to transcribe genes critical for cell cycle progression. CDK4 and CDK6 are inhibited by members of both the inhibitors of CDK4 (INK4) and CDK interacting protein/kinase inhibitory protein (Cip/Kip) family of CDKIs. Targets of the E2F transcription factors include cyclin A and cyclin E which are required in conjunction with CDK2 late in the G₁ phase. CDK2 is inhibited by members of the Cip/Kip family of CDKIs, and also reinforces the inactivation of RB by maintaining its phosphorylation, and completing the transition into the S phase (Lapenna & Giordano 2009). Mixed-lineage leukaemia (MLL) family members and menin activate transcription of p18^Ink4c and p27^Kip1 (Milne et al. 2005). Red arrows indicate activation/up-regulation, blue lines indicate inactivation; red boxes/text indicate active proteins and blue boxes/text indicate inactive proteins.

• © 2011 Society for Endocrinology