24-12-2012, 05:52 PM
Transgenic Animals: A Focus on Transgenic Mice Studies
Transgenic Animals.ppt (Size: 1.23 MB / Downloads: 75)
Introduction
Transgenic animals:
Animals which have been genetically engineered to contain one or more genes from an exogenous source.
Transgenes are integrated into the genome.
Transgenes can be transmitted through the germline to progeny.
First transgenic animal produced = “Founder Animal”
Introduction of foreign genes into intact organisms
Procedure is basically the same regardless of which animal is involved.
Integration usually occurs prior to DNA replication in the fertilized oocyte.
Majority of transgenic animals carry the gene in all of their cells, including the germ cells. Transmission to next generation requires germline integration.
Some integration events occur subsequent to DNA replication giving rise to mosaic animals which may or may not contain the transgene in its germline.
Procedure for Producing Transgenic Mice
Three different breeding pairs of mice are required.
First Breeding Pair:
Fertile male + superovulated female
Fertile male = stud (changed regularly to ensure performance)
Superovulated female = immature female induced to superovulate
Pregnant mare’s serum (=FSH) on day 1
Human Chorionic Gonadotropin (=LH) on day 3
Mated on day 3
Fertilized oocytes microinjected on day 4 with foreign DNA construct.
Microinjected oocytes are transferred to the oviducts of surrogate mothers at end of day 4.
Integration of Transgene into One Chromosome
Normally the transgene inserts into one chromosome giving rise to a heterozygote.
50% probability of passing transgene onto offspring.
Two heterozygous mice may be bred to obtain a homozygous line that contains the transgene on both chromosomes.
100% probability of passing transgene onto offspring.
Most transgenes are stably transmitted for many generations without detectable rearrangement.
Mechanisms of DNA Integration
Linear molecules integrate more efficiently than circular molecules (~5x)
Once in the oocyte, the linear molecules circularize.
Usually all of the molecules that integrate are on the same chromosome and at the same site.
Multiple copies are usually arranged in a tandem, head-to-tail array.
The size of the DNA molecule (0.7 – 50Kb) is not an important parameter.
The concentration and purity of the injected DNA is critical (1-3 mg/ml maximum).
Working Hypothesis of DNA Integration
The ends of the injected linear DNA integrate at breaks that occur spontaneously in the chromosome.
Other injected molecules which have circularized probably recombine with each other and the integrated copies to generate a tandem, head-to-tail array.
Recombination is probably favored because of high local DNA concentration and special properties such as the absence of normal chromatin structure.
The number of chromosomal breaks is presumably limiting explaining the low number of integration events and why different DNA molecules are usually integrated at the same site.
Gene Expression in Transgenic Mice
In order to discriminate the products of the injected gene from those of an endogenous counterpart, the injected gene must be marked in some way.
Mini-genes where exons are deleted of cDNA where introns are absent.
Modification by insertion/deletion/mutagenesis of a few nucleotides (e.g. the gain or loss of a restriction endonuclease site).
Hybrid genes where foreign epitopes are expressed on transgenic products.
Tissue-Specific Gene Expression
Generally, if a tissue-specific gene is expressed at all, then it is expressed appropriately, despite the fact that it has integrated at a different chromosomal location.
Trans-acting proteins involved in establishing tissue-specific expression are capable of finding their cognate sequences and activation transcription at various chromosomal locations.
Levels of expression vary between founder animals as chromosomal position can influence accessibility of the transgenes to these trans-acting transcription factors.
Some founders do not express the transgene at all owing to integration into heterochromatin domains where DNA is methylated heavily (silent).