(ES) cell lines which, after manipulation and selection ex vivo, can then be introduced into early embryos, some of whose germline will develop from the ES cells (Smith, 2001); and (5) manipulation of cultured somatic cells, whose nuclei then can be transferred into enucleated oocytes and thereby provide the genetic information required to produce a whole animal. The last two methods have the advantage of allowing cells containing the modification of interest to be selected prior to undertaking the expensive and lengthy process of generating animals. Usable ES cells are not available for all species of interest, however, and generation of embryos by nuclear transfer (NT) from somatic cells is becoming the method of choice for genetic engineering and duplication of nearly genetically identical animals (Westhusin et al., 2001).
Manipulation of the avian germline is difficult since ES lines are not available and the early embryo is difficult to access. Much current work focuses on the use of blastodermal cells or primordial germ cells, which can be cultured briefly and manipulated to modify the germline prior to introduction into fresh embryos to create chimeras from which modified lines can eventually be developed, albeit with low efficiency (Aritomi and Fujihara, 2000).
There are two basic approaches presently in use for inserting DNA into vertebrate germline cells, transfection and infection with retrovirus vectors. A third approach, based on the use of mobile genetic elements, has been commonly used for insects and is being explored for germline modification of vertebrates (Izsvak et al., 2000).
Transfection methods include: (1) direct microinjection of DNA into the cell nucleus; (2) electroporation—introduction of DNA through transient pores created by controlled electrical pulses; (3) use of polycations to neutralize charges on DNA and the cell surface that prevent efficient uptake of DNA; (4) lipofection, or enclosure of DNA in lipid vesicles that enter a cell by membrane fusion much in the manner of a virus, and (5) sperm-mediated transfection, possibly in conjunction with intracytoplasmic sperm injection (ICSI) or electroporation (see Chapter 6). The manner of introduction of DNA is a technical issue, determined empirically for each system, and makes little difference to the final outcome. In general (with the exception of homologous recombination, discussed below), the structure of DNA introduced into a cell by any of these methods is highly variable and uncertain. Often, only a fragment of the transfected DNA is integrated into the chromosome, frequently in multiple copies, that often are integrated in long tandem arrays (Gordon and Ruddle, 1985). When transfecting cultured somatic or ES cells, a selectable marker, such as the gene encoding phosphotransferase, is often included as part