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by the transcription of areporter genesuch as theCATgene. The technique is not just

confined to transcription factors and may be applied to any protein system where

interaction occurs.

6.8.4 Transgenics and gene targeting

In many cases it is desirable to analyse the effect of certain genes and proteins in an

organism rather than in the laboratory. Furthermore the production of pharmaceutical

products and therapeutic proteins is also desirable in a whole organism. This also has

important consequences for the biotechnology and agricultural industry (Section 6.10)

(Table 6.4). The introductionof foreign genesinto germline cells andthe production of an

altered organism is termedtransgenics. There are two broad strategies for transgenesis.

The first isdirect transgenesisin mammals whereby recombinant DNA is injected

directly into the male pronucleus of a recently fertilised egg. This is then raised in a

foster mother animal resulting in an offspring that is all transgenic.Selective transgen-

esisis where the recombinant DNA is transferred intoembryo stem(ES) cells. The cells

are then cultured in the laboratory and those expressing the desired protein selected and

incorporated into the inner cell mass of an early embryo. The resulting transgenic animal

is raised in a foster mother but in this case the transgenic animal is a mosaic or chimeric

since only a small proportion of the cells will be expressing the protein. The initial

problem with both approaches is the random nature of the integration of the recombinant

DNA into the genome of the egg or embryo stem cells. This may produce proteins in cells

where it is not required or disrupt genes necessary for correct growth and development.

A refinement of this however isgene targetingwhich involves the production of an

altered gene in an intact cell, a form ofin vivomutagenesis as opposed toin vitro

mutagenesis (Section 6.6.2). The gene is inserted into the genome of, for example, an

ES cell by specialised viral-based vectors. The insertion is non-random, however, since

homologous sequences exist on the vector to the gene and on the gene to be targeted. Thus,

homologous recombinationmay introduce a new genetic property to the cell, or inacti-

vate an already existing one, termedgene knockout. Perhaps the most important aspect

Table 6.4Use of transgenic mice for investigation of selected human disorders

Gene/protein Genetic lesion Disorder in humans

Tyrosine kinase (TK) Constitutive expression of gene Cardiac hypertrophy

HIV transactivator Expression of HIVtatgene Kaposi’s sarcoma

Angiotensinogen Expression of rat angiotensinogen gene Hypertension

Cholesterol ester transfer
protein (CET protein)

Expression ofCETgene Atherosclerosis

Hypoxanthine-guanine
phosphoribosyl transferase
(HPRT)

Inactivation ofHPRTgene HPRT deficiency

248 Recombinant DNA and genetic analysis