Purification Of Transformant

Purification of the transformed nucleus is facilitated in those fungi that form uninucleate conidia—for example, Aspergillus (Chapter 7)—or that can be manipulated to selectively produce uninucleate cells, as with N. crassa (Maheshwari, 2000). In N. crassa, macroconidia are easily obtained and are therefore routinely used for transformation. However, as these cells are usually multinucleate, the transforming DNA integrates at random locations in the genome and in different numbers of copies in different nuclei. Since not all nuclei in the same cell are transformed similarly (Pandit and Russo, 1992; Groteleuschen and Metzenberg, 1995), the transformants are commonly heterokaryotic. Thus, a single transformed nuclear type needs to be purified. During macroconidia formation, nuclei from the mycelium enter into macroconidia in varying numbers, generally between one and four. The primary transformants are heterokaryotic, which requires the purification of a transformant having a single nuclear type. This is done by the rather time-consuming method of repeatedly plating a dilute suspension of macroconidia in series and picking up single colonies showing the transformed phenotype. Alternatively, a genetic method can be used to purify a transformed nucleus from a heterokaryotic transformant by crossing it to an untransformed strain and selecting the transgene-bearing segregants among the meiotic progeny. However, purification of the transformed nucleus by the crossing method has generally been unsuccessful because very often the transgene is not transmitted to the progeny.

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