Michalovich, David; (1995) Homologous recombination in embryonic stem cells: Targeting of the murine Thy-1.2 and Zfp 37 genes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Establishment of the central nervous system follows a complex pathway of cell-to-cell interactions and sequential gene expression. Interest in our lab has focused on two genes showing neuronal expression. The Thy-1 gene encodes an immunoglobulin-like cell surface glycoprotein which is thought to function as a receptor/adhesion molecule involved in the cessation of axonal growth. The murine Zfp-37 gene is a member of the Krüppel-associated box (KRAB) Cys2His2 zinc-finger family of genes and is postulated to play a role in transcriptional regulation. To address the in-vivo functions of these genes a targeting approach in mouse embryonic stem cells (ES) has been followed to produce "loss of function" mutants. An ES cell clone carrying a targeted Thy-1 allele was successfully obtained, however this failed to give germline transmission. Mice carrying a mutated Thy-1 allele became available from a another laboratory and therefore the project was pursued as a collaboration. The murine Zfp-37 gene was disrupted by insertion of a lacZ reporter gene via homologous recombination in ES cells. On a three strain genetic background homozygous Zfp-37-/- mice appear healthy and fertile and are represented at close to the predicted Mendelian ratio. On a (129 x C57BL/6) genetic background three of the six homozygous animals obtained have died, however, it remains to be determined if the deaths are linked to the targeted mutation. The targeted reporter gene expression has shown Zfp-37 to be consistently expressed in the developing nervous system of the embryo from embryonic day 9.5 to 12.5. Other areas of the embryo also show expression such as the hind gut (e9.5) and the developing limbs (e10.5-e12.5). In the three week postnatal brain high levels of reporter gene expression are found in the hippocampus, superior colliculus, hypothalamus, olfactory tuberculum and granular and Purkinje cell layers of the cerebellum. Analysis of the neuronal reporter gene expression in Zfp-37+/- and Zfp-37-/- mice suggest ZFP-37 to be part of an autoregulatory mechanism regulating its own expression.

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