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Transgenes

A transgene is a gene randomly inserted into a host genome (the transgenic organism). Transgenics (aka random integration or non-targeted transformation) is the usage of transgenes in research. A lineage of transgenic animals is created in the steps described below. If the transgene is not phenotypically obvious, then PCR analysis can be used to verify its presence. Also, a transgene should include: a promoter so that it is expressed; an enhancer to help bind RNAP; intronic sequences, to emulate a typical eukaryotic genome; and a polyadenylation [poly(A)] sequence to ensure mRNA stability. The promoter can be inducible, meaning it requires a foreign agent to activate it.

A common inducible promoter is the metallothionein promoter, which is inducible by heavy metal ions. Another heavily used transgene promoter is repressed by a tet-controlled transactivator (tTA) and viral activation domain (HSV VP16) dimer which binds and prevents RNAP form binding. However, tetracycline injected into the animal will bind this binder and keep it from inactivating the promoter, thus allowing the transgene to be expressed. Lastly, other promoters are only activated within certain tissues (such as liver, kidney or other tissues).

There are unpredictable factors which influence transgene expression. For example, multiple copies of the transgene might integrate into the host genome, thus increasing transgene expression. Another possibility is that the transgene will integrate downstream from an enhancer (resulting in high expression) or in a completely inactive region of the genome (leading to reduced expression).

FertilizationA stud male and super-ovulating female mate. The resulting litter of fertilized eggs (oocytes) is isolated at the pronuclear stage, when male and female pronuclei are still separate and distinguishable.
MicroinjectionPicoleters of transgene DNA are microinjected (with a very fine hollow needle) into the male pronuclei of the oocytes. The exogenous DNA integrates into male pronuclei DNA upon chromosomal decondensation.
BackcrossingBackcrossing verifies that the transgene was integrated into the genome and is not just expressed independently. A transgenic animal is mated (backcrossed) with a non-transgenic animal: if any offspring express the transgene, then the DNA was integrated; if no offspring express the transgene, then the DNA was not integrated.

Transgenes are used extensively, encoding everything from growth hormone (resulting in huge mice) to green fluorescent protein (allowing for better tumor detection when engineered to express only in tumor cells). At this point, please read about gene targeting if you are unfamiliar. What are some uses of transgenic animals in developmental biology? A DNA construct can be engineered with a gene for Organ A attached to a promoter for Organ B; the transgenic animal is observed for whether Organ A is now formed in the region of Organ B. Alternatively, a DNA construct can be engineered with a dominant negative mutation. The mutant gene product interferes with the wild-type gene product to cause loss of function. (Developmental Biology Genetics Laboratory Techniques) (Transgenic Animals)

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