Animal models of pituitary neoplasia

Abstract

Pituitary neoplasias can occur as part of a complex inherited disorder, or more commonly as sporadic (non-familial) disease. Studies of the molecular and genetic mechanisms causing such pituitary tumours have identified dysregulation of >35 genes, with many revealed by studies in mice, rats and zebrafish. Strategies used to generate these animal models have included gene knockout, gene knockin and transgenic over-expression, as well as chemical mutagenesis and drug induction. These animal models provide an important resource for investigation of tissue-specific tumourigenic mechanisms, and evaluations of novel therapies, illustrated by studies into multiple endocrine neoplasia type 1 (MEN1), a hereditary syndrome in which ∼ 30% of patients develop pituitary adenomas. This review describes animal models of pituitary neoplasia that have been generated, together with some recent advances in gene editing technologies, and an illustration of the use of the Men1 mouse as a pre clinical model for evaluating novel therapies.

Keywords: Adenoma; Carcinoma; Mouse; Multiple endocrine neoplasia type 1; Pituitary; Rat.

Fig. 1

Gene knockout methods using embryonic stem (ES) cells. A Totipotent ES cells are isolated from the inner cell mass of a blastocyst from a wild type mouse and cultured. B Targeted or non-targeted vectors are introduced into the genome of the ES cells. ES cells in which homologous recombination or random integration and i.e. gene knockout, has occurred are selected using incorporated markers (e.g. Neo). C Selected ES cells are injected into a blastocyst obtained from a different strain to that in step A, and implanted into a pseudopregnant female, which is the same strain as the injected blastocyst. This results in chimeric offspring containing genetic information from both the manipulated ES cells of the original mouse strain and genetic information of the second blastocyst/pseudopregnant female of a different strain. To generate offspring heterozygous for the gene knockout, chimeric offspring are bred with wild type mice. D Heterozygous offspring can be interbred to generate homozygous knockout mice. m-mutated allele; + – wild type allele.

Fig. 2

Conditional gene knockout. Gene knockout models can be generated using the FLP-Frt or Cre-LoxP systems. This requires the generation of two constructs: 1) a construct containing Frt or LoxP recognition sites inserted into the intron sequences flanking the genomic region to be knocked out; and 2) a construct containing a FLP or Cre recombinase under the control of a tissue-specific promoter. These constructs are introduced into two different mouse strains using knockin/transgenic over-expression methods, to generate one mouse expressing the Frt/LoxP flanked genomic sequence in all tissues, and one mouse expressing FLP/Cre recombinase in a specific organ e.g. pituitary. These two mouse lines are then bred to generate a mouse containing both the Frt/LoxP flanked genomic sequence and the tissue-specific recombinase. In FLP/Cre-expressing tissues e.g. pituitary, the FLP/Cre binds to its target Frt/LoxP sites and catalyses recombination of the DNA, leading to excision of the genetic material contained between the target sites.

Fig. 3

N-ethyl-N-nitrosourea (ENU) mutagenesis. ENU is a chemical mutagen that induces point mutations via transfer of its ethyl group (CH₃) to guanine residues, which in turn causes mispairing during DNA replication. ENU is injected into male mice where it causes mispairing during spermatogenesis, and therefore induces mutations into sperm DNA. Mutagenised mice are mated with female mice of the same strain to generate offspring that have inherited the introduced mutations. Genetic and phenotypic screens are performed to characterise the mutation. m-mutated allele; +–wild type allele.

Fig. 4

Gene editing using CRISPR/Cas. The CRIPSR/Cas system requires three components: a CRISPR-associated nuclease, for example Cas9; a single guide RNA (SgRNA) consisting of a guide sequence that binds the target DNA, a scaffold sequence for Cas9 binding and a terminating hairpin; and for gene knockin an oligo template containing the sequence to be inserted. The Cas9 is targeted to a specific genomic site by the SgRNA, where it induces a double strand break. In the absence of a repair template this break is repaired by non-homologous end joining, an error prone mechanism that leads to insertion/deletion (indel) mutations, which in turn cause frameshifts and the occurrence of premature stop codons, thereby leading to gene knockouts. If a DNA repair template is present the double strand break is repaired by a homology-driven repair mechanism, using this template, therefore genetic information can be knocked-in by including, for example, gain-of-function point mutations in the inserted template.