Creating a transgenic animal
A transgene is isolated, and using a microinjection it is inserted into an egg just after it has been fertilised. Because it is done before the nuclei from the egg and sperm have fused, the transgene can be injected into the larger sperm nucleus (an easier task). For a few divisions the embryo is left in the cell culture and then it is transferred to the womb of a foster mother. If successful, the foster mother will birth a founder animal which has the transgene. This founder animal is mated to another founder animal, 25% of the children will be homozygous for the transgene. These animals are useful because further inbreeding means they will all have the transgene. Typically a male is preferred because a male can mate to multiple females quickly.
If several cell divisions occur before successful integration of the transgene, the animal will be chimeric because the variability in transgene uptake in cells at the embryo level is amplified to all cells when the animal is fully grown. Another issue is that the gene might integrate somewhere which isn’t expressed (methylated DNA?) or might knock out another gene by integrating into it. Often multiple copies are inserted in tandem, this can be dealt with by using a retrovirus instead of a microinjection.
Rat growth hormone in mice
The gene for the polypeptide somatotropin was cloned from rats into fertilised mouse eggs. The mice which were born of the foster mother were larger due to them producing the growth hormone. The promoter used was from the metallothionein gene. This promoter is active in the liver, and so while growth hormones are usually made in the pituitary gland, this growth hormone was made in the liver.
Recombinant protein production in livestock
Because cows produce so much milk, and because expressing recombinant proteins in bacteria requires a skilled workforce, it may be advantageous to express these proteins in livestock milk. Recombinant cows have the cloned gene for the protein of interest inserted with a promoter such that it will only be expressed in the mammary glands of the animal. The gene usually replaces the beta-casein gene, which is under the control of the endogenous promoter.
Goats are used to make rTPA nowadays.
We can discover the functions of genes by knocking them out and observing changes in mice because many genes have no obvious phenotype. One first must clone the gene to be knocked out, and then insert a DNA cassette into the sequence, which breaks the gene function. The gene can then be inserted back into the animal as discussed previously and sometimes the DNA will integrate into genome and replace the original gene due to homologous recombination. These mice can be bred together to produce offspring with both copies of the KO gene to produce KO mice. The phenotypic changes can then be observed (if they survive).
Embryonic stem cells for genetic engineering
Retroviruses can be used to infect early embryos. Only a single copy of the gene is inserted and one does not need to be skilled in microinjection techniques. Because the DNA is not incorporated at the exact time that the nuclei fuse however, founder animals are born chimeric. This is an advantage in some cases because normal tissue and KO/modified tissue can be compared directly in the same animal. Also if the transgene incorporates into cells which end up becoming the germ-line, then these chimeric animals can be bred to create fully engineered offspring.
Embryonic stem cells can be taken from the blastocyst of a developing mouse and cultured under conditions which do not stimulate differentiation. Transgenes are introduced into the stem cells, (often with a fur colour marker) and then the stem-cells are inserted back into the central cavity of the developing embryo. This produces a chimeric mouse and one can determine if the experiment was successful by identifying if there is a patchwork of different fur colours on the newly born mice. If the newly born mice have the engineered stem-cells forming germ-line cells, then they can be bred together to form fully transgenic mice.
The location where the transgene gets incorporated into the genome affects the expression level of that gene in the animal. This leads to high variability in different animals with the same transgene. Recall that enhancer sequences may work on genes which are thousands of base pairs away, and other regulatory elements around the gene will affect expression. If the gene is inserted into the heterochromatin then it will not be expressed at all, or very little.
One can insert the transgene with its own enhancer sequences. For example a locus control region can be cloned in with the gene which leads to high expression and typically “overpowers” other nearby regulatory elements. The LCR is placed in front of the gene to be expressed.
Insulator sequences can be used which block other control elements in the region from affecting the expression of the gene. One can clone in the gene with its regulatory sequences and flank the whole thing with insulator sequences which will prevent any nearby regulatory elements from changing expression.
It has been found that a full length transgene, including the introns are not affected as much by position effects. Therefore by cloning in the full gene instead of just cDNA one has a higher chance of negating position effects, however this is more difficult due to having to handle longer pieces of DNA.
homologous recombination can be used to target the transgene to a specific location in the genome. This is good if the transgene to be inserted is just a version of an existing gene, and so inserting it into the same location as the native gene means that it will be expressed at the same rates with the same regulation. Targeting vectors are the transgene flanked by regions which are homologous to the section of the genome you want to clone into. With targeting vectors one can insert or replace DNA (figure 16.9). Linearising DNA stimulated recombination.
Promoters such as the metallothionein promoter (which requires toxic zinc) and the heat shock promoter are not very practical for constant induction of protein expression. Recombinant promoter systems are better.
The tet operon can be used to control expression in transgenic animals. TetR has been fused to eukaryotic transcription factor VP16. This means that TetR will recognise the tetO site, but becuse it’s bound to a transcription factor, it actually activates transcription. Therefore when tetracycline is added, the TetR will unbind and the gene will be repressed.
The opposite can be made true using a mutant TetR protein which is only able to bind to DNA in the presene of tetracycline, this makes it so that tetracycline induces the gene to be activated. It is called the reverse tet transactivator system (16.12).
Site specific recombination
Use of the Cre/loxP system has already been discussed in L15. It can be used in transgenic animals to remove selective markers after successful integration of DNA into the genome as they are no longer needed. It can be used to activate a transgene if it has been inactivated by adding blocking sequences to it. By doing two rounds of insertion of loxP sites at different locations one can use Cre recombinase to delete huge amounts of DNA.
The recombinase system can be used much like it is in plants. Two separate mice can be engineered, one with the recombinase system and one with the transgene and loxP sites. When mated, the deletions occur in their offspring.
Integrase is a single protein which can insert DNA into the genome. The DNA to be integrated and the host genome need to have att sites present on both (attB and attP respectively). This is not dissimilar to the GATEWAY cloning system.
DNA inserted into the germ-line of mosquitoes can enable them to carry a “genetic suicide system”. If one inserts two genes, A and B, which both must exist in the organism for survival, are cloned into males, which are then released into the wild, then engineered-male and wild-female will mate, and their hybrid offspring will survive. When hybrids breed however, some of them will only get either A or B, meaning they will have fewer offspring. And subsequent generations will progressively get smaller and smaller. This could in theory wipe out a population of mosquitoes.
References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.