（二)基因的转运

　　目前已有多种基因转运的方式，其基本原则是将外源基因运到细胞内。已使用的有病毒载体和非病毒载体两大类。

　　病毒载体：病毒具有一些独特的性质如多数病毒可感染特异的细胞，在细胞内不易降解；RNA病毒能整合到染色体以及基因水平较高等。因此病毒载体是良好的基因转运载体。目前已被用作载体的病毒有逆转录病毒、腺病毒、腺相关的病毒。疱疹病毒和肝炎病毒等。逆转录病毒用作载体时需进行几步改造：（1)将天然的野生型RNA前病毒转变成DNA载体，并插入欲转移的相关外源基因。其基本原则是用标记基因和外源基因替代病毒的编码基因，如图23-2所示。（2)制备辅助细胞为载体DNA提供其丧失的功能，如图23-3所示。（3)将载体DNA导入辅助以产生病毒载体。如图23-4。（4)病毒载体感染靶细胞，外源基因在细胞内得以表达，如图23-5所示。

STEP 1

INSERTING FOREIGN DNA INTO THE RETROVIRAL PROVIRUS

Wild-Type Provirus

图23-2 Scientists use recombinant DNA techniques to replace the gag,pol,and env genes with one or more foreign genes.The foreign genes can be inserted in several different patterns.in this example,a selectable marker gene(neo)replaces the viral gag and pol genes and　a human gene replaces the env gene.

STEP 2

MAKING THE HELPER CELL

Desiging the Helper Vius

图 23-3 The helper cell should meet two basic requirements:(1)it should provide functions missing from the the vector virus,and(2)it should not be capable of producing viable virus particles. The crucial element in the development of a successful helper cell is the design of the helper virus.Researchers use recombinant DNA techniques to disable the helper virus in the test tube-one of the most common measures is removal of the Psi fragment.The Psi-deficient helper virus produces all of the normal viral proteins, but cannot package its own RNA because it lacks the appropriate packaging signal. Helper virus DNA is inserted into the genome of the helper cell using chemical techniques.

STEP 3

PRODUCING THE VECTOR

图23-4 Scientists use chemical measures or an infection technique to insert recombinant vector DNA (including a human gene) into helper cells.Because the vector provirus contains the Psi sequence, the vector RNA genome is automatically encapsulated by viral proteins produced by helper virus DNA in the helper cell. The resulting viral particles are released by budding from the helper cell membrane. The vector virus is capable of only one infection because it lacks the information needed to make viral proteins.

STEP 4

INFECTING THE TARGET ELL

图23-5 Researchers infect target cells (such as human bone marrow cells)with the vector virus in two different ways: they mix them with helper cells producing the virus, or they bathe them in fluid harvested from the helper cell culture.When the vector provirus is integrated into the target cell DNA ,enzymes from the target cell treat it as an integral part of the genome. Cellular enzymes do the work necessary to make proteins from the foreign genes located between the two viral L TRs.

　　非病毒载体：这类载体的发展较快，目前主要是脂质体，一些具有受体功能的载体也呈现出诱人的前景，关于常见载体的优缺点列于表2。

表23－2　常见基因转运载体的优缺点

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