7 
Dt. Scanqos said transgenic animals are made for several reasons: (1) to study 
tissue and staqe-specific gene expression in development; (2) to study phenotypic 
effects of foreiqn gene expression; (3) to study oncogene expression; (4) to 
study insert ional mutaqenesis; and (5) to produce animal models of human 
disease. In the future, a sixth reason may be to create transgenic "factory" 
animals which would produce medically important compounds. 
Dr. Scanqos said there are four methods of introducing foreign DNA into animals: 
(1) microinjection of foreiqn DNA into the blastocoel cavity of early embryos; 
(2) exposure of early embryos to an infectious retrovirus; (3) transfecting 
DNA into totipotent teratocarcinama cells followed by injection of selected 
cells into the blastocoel; alternatively, nuclei from such cells can be 
introduced into fertilized egqs fron which the pronuclei have been removed; 
and (4) microinjection of DNA directly into the pronuclei of fertilized eggs. 
Dr. Scanqos said microinjection of DNA into the pronuclei of fertilized eggs 
is the technique most used for introducing foreign DNA. Microinjection allows 
a variety of different DNA molecules to be introduced into the cells of the 
experimental animals; there is no constraint on the size or the sequence of 
the DNA to be introduced. Superovulated female mice are mated, and the one-cell 
embryos of the pronuclear stage are removed from the reproductive tract. The 
one-cell embryos are stabilized against a blunt-end glass pipette and foreign 
DNA injected throuqh a fine glass needle into one of the two pronuclei. Approxi- 
mately 50% of the injected embryos are lost at this point. The surviving 
microiniected embryos are then implanted in the oviducts of pseudopregnant 
female mice. Tan to 20% of the implanted embryos develop into mice. Twenty-five 
percent of these offspring will have incorporated the foreign gene(s) into 
their genomes. A subset of the animals incorporating the qene into their 
chromosomes express the foreiqn gene. The success rate of this technique in 
obtaining mice expressing the foreign qene is approximately 1 to 3% depending 
on the season and the mouse strain. 
Dr. Scanaos said the injected DNA most often integrates into a single chromosomal 
site; the site of insertion cannot be controlled experimentally. Multiple 
copies of the injected DNA are typically arranged in the chromosomal insertion 
site in tandem head-to-tail arrays containing up to several hundred copies; 
copy number is not under experimental control. The mechanism of integration 
is not known; the foreiqn DNA might be incorporated either by recombination or 
durinq chromosome repair. Microinjected DNA appears to integrate randomly at 
any site in the genome including the middle of genes. How multiple copies of 
the microinjected DNA integrate into the chromosome at a single site is not 
known. Since multiple copies of the foreign gene are injected into the embryo, 
one possibility is that one copy of the injected gene may integrate into a 
chromosomal site; and other copies may subsequently integrate by homologous 
recombination with the original copy. The other possibility is that long 
chains of copies of the injected gene form in the cell and subsequently integrate 
into a single chromosomal site. 
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