San Ling SiHoe and David Murphy 1 Introduction

A pioneering experiment in the early 1980s demonstrated that microinjection of recombinant growth hormone into the pronuclei of fertilized one-celled mouse embryos resulted in inheritable changes in the growth of these mice (1). Mammalian transgenic experiments have since contributed tremendously to our understanding of numerous complex biological processes. The power of the technique lies in that it allows the function, and developmental and physiological regulation of almost any protein to be studied within the context of the normal processes occurring in the whole animal.

It is unknown how DNA integrates into the host chromosome, but some information can be inferred from a study made on the state and organization of the inserts found in transgenic mice (2). Approximately 70% of the mice carry exogenous DNA in all their somatic and germ cells, implying that integration took place prior to the first round of DNA replication. The remaining 30% of the mice showed some degree of mosaicism, indicating that the DNA must have integrated after this first round of replication. Integration events have been observed on many different autosomes (3), on the X-chromosome (4), and on the Y-chromosome (Murphy, unpublished observations). Transgene copy number varies (one to several hundred), but within a transgenic animal, there is usually only one integration site where if in multiple copies, the transgene is arranged in a head-to-tail tandem array.

Once incorporated in the DNA, the transgene transmits in a classic Mende-lian fashion. Appropriate tissue-specific and physiologically regulated expres-

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

sion of the transgene is a prerequisite in many transgenic experiments, and can only be obtained by including the appropriate genetic elements in the structure. Once appropriate expression is obtained, gain-of-function (overexpressing the gene of interest) or loss-of-function (cell-specific expression of cytotoxic proteins) approaches can be employed to derive information about almost any gene or cell type. Studies of the nervous and immune systems and oncogenesis (5-7) are some examples where transgenic animal studies have contributed significantly to our knowledge.

This chapter details the procedures necessary for the generation of transgenic mice and rats by the injection of cloned DNA into the pronuclei of fertilized one-celled eggs. The technique demands precise technical skill and expensive equipment, but its reliability and speed make it currently the most efficient way of generating transgenic mice. It is still the only route of making transgenic rats with inheritable genetic alterations. There are other methodologies, such as the infection of preimplantation embryos with retroviruses or homologous recombination in embryonic stem cells.

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