Lecture 21

Somatic cell genetics and transgenic animals



A word on getting started...

With many genetically-tractable organisms fully or partially sequenced, it is easy to be drawn towards expression systems that are well established. Suppose, however, that you are working on an organism for which you have little information. You don't have an example of a promoter, and know little about how to introduce DNA. How would you get started?

Here is what you will need for this "expedition" into the frontiers of science:

  • A method to introduce DNA (i.e. calcium phosphate, lipofectins, electroporation, gene guns, etc.).
  • A means of determining whether the DNA has been taken up and expressed (i.e. a "reporter" gene)


Once DNA has entered a cell, in rare cases the DNA will make its way to the nucleus and be expressed, provided that it contains the necessary regulatory sequences (for example, a promoter for RNA polymerase). Since these regulatory sequences are not always functionally conserved, meaning that a promoter sequence active in one organism (or tissue) may be inactive in another. Sometimes you have little choice but to try first to learn about gene regulation in your organism, a process that may take years. You must determine where transcription starts for a highly expressed gene,



and hope that sequences surrounding the mRNA start site are sufficient for transcription initiation in a new context.

PLAN ONE: Transient transfection of cells.

The way to find out if your DNA is being expressed is to introduce a "reporter gene," which encodes an easily assayed gene product, into your plasmid.


Now, you simply play with the knobs on your electroporation machine, trying to get the conditions just right so that DNA is taken up by the cells. Reporter gene expression is your only assay! Even if your DNA is taken up by cells, you also don't know whether most of your reporter gene will be expressed 5 hours after transfection or 50 hours after transfection. This type of work will lead you to many nights with little sleep, but you may eventually succeed in learning how to introduce DNA into cells of your favorite organism and have the DNA be expressed.

PLAN TWO: Stable transfection of cells

Once you realize that your transient transfections are not going too well, and you've just wasted half a year of your life, you may start to think that your favorite organism doesn't deserve to live! This can be arranged.

Eukaryotic cells tend to be sensitive to some antibiotics such as G418 (a derivative of Neomycin) and hygromycin, for which resistance genes are available. You can use your cloning techniques to place a resistance gene into the DNA from your highly transcribed region (deleting the native gene) and introduce that linearized DNA into cells, promoting homologous recombination.




The end result is that a drug resistance gene can be integrated into the genome, in place of a native gene:



Now if you grow your transfected cells in hygromycin, cells that are not expressing the Hyg resistance gene will be killed by the drug. Some of the cells that are hygromycin resistant may have integrated the gene by homologous recombination (as shown above) and others may have integrated it into another chromosomal locus.

Joining the crowd

Of course it is far easier to work with an organism that has already been well characterized, such as yeast. There are many yeast vectors available that permit integration (the YIp type) or maintenance as an extrachromosomal element (the YEp type, which have origin of replication elements). Using these vectors, beautiful and elegant genetic experiments can be performed. An example is the "plasmid shuffle" experiment:

Let's suppose you've used your genetic engineering skills to make a mutated allele A for a gene of interest, and you want to introduce it into cells and have it be the only copy of the gene. If the gene is required for viability, how will you exchange the wild type for the A allele without killing the cell?


By this method, one plasmid has been exchanged for another, and consequently one allele of a gene has been exchanged for another. Very slick genetics!

On the other hand, before you leap onto the yeast bandwagon and take this well traveled road, you might want to consider that there are more yeast researchers than yeast genes!

Homologous and non-homologous recombination

How can you test for homologous recombination? Offer the cell several TK genes on the side, and select against cells that incorporate them. Homologous recombination events that exclude the TK genes are preserved.





More about that later - but first, let us talk about how to get from cell line to animal.
Transgenic animals

Understanding disease processes, gene function, and development

Why make animals transgenic? With the ability to knock out (i.e. destroy) or modify individual genes in an animal, we have a tremendous potential to understand the relationship between genes and development, and the causes of genetic diseases.

We can also develop animal models for human diseases, such as cystic fibrosis, sickle cell anemia, and AIDS. An interesting sites to visit is:

The transgenic and targeted mutant animal database

and for the sake of art:

Green fluorescent mouse: http://www.mshri.on.ca/nagy/cre.htm

Down on the pharm

"Pharming" is a term used to describe the production of human pharmaceuticals in animals. As an example, transgenic mice have been used to produce a human drug, tPA (tissue plasminogen activator to treat blood clots) since 1987. This type of approach for producing pharmaceuticals is particularly useful when the product is modified (glycosylated, etc.), and correct modification is not feasible in an in vitro system. A popular method of "pharming" is to couple the DNA gene for the protein drug with a DNA signal directing production in the mammary gland. The foreign protein is expressed and secreted into the milk (of a goat or cow, for example) and doesn't interfere with the health of the animal. A goat expressing tPA in its milk is worth an estimated $75,000 per year. A pig expressing human protein C (another clot-busting drug) is worth about $1,000,000 per year. Pig-napping may become a more common crime, as the pharming develops.


Reference article on pharming

Animals need not be developed with a specific product in mind. Imagine having a farm animal with an entirely humanized immuoglobulin locus. The animal would produce, from birth, humanized antibodies to any immunogen. Monoclonal antibodies generated from such an animal would be an ideal pharmaceutical agent.

How to do it...by stem cell methods


http://www.ultranet.com/~jkimball/BiologyPages/T/Transgenics.gif

Transfer of Embryonic Stem Cells
- Injection of ES cells into the cavity of blastocysts using a microscope (inverted), micromanipulator equipment and transfer / holding devices [2].

Embryonic stem cells are derived from the inner cell masses of normal blastocysts (early embryos, mostly mouse embryos). These cells are pluripotent, which means they can develop into any type of tissue. Therefore, removing ES cells from the culture and placing them back into an early embryo (blastocyst) allows them to divide and become part of the embryo. Transfection allows foreign genetic material to be inserted in vitro into these cells. The target of this process is homologous recombination with the chromosome of the cell, i.e. introduction or exchange of DNA at one location with homologous DNA sequences. Following transfection, the descendants of one individual cell (clone) are analyzed using microbiological methods to ascertain whether any mutation is present. Cells of the identified clones which showed the desired reaction are multiplied in vitro and injected into blastocysts. 

These injected blastocysts are then implanted into the uterus of pseudo-pregnant females.

How to do it...by microinjection

Microinjection of DNA

Injection of linear DNA molecules into fertilized eggs (pronuclear stage) using an inverted microscope, micromanipulation equipment and injection / holding devices.

The first successful production of transgenic mice using pronuclear microinjection was reported in 1980 [1]. The pronuclear microinjection method of producing a transgenic animal results in the introduction of linear DNA sequences into the chromosomes of the fertilized eggs. If this transferred genetic material is integrated into one of the embryonic chromosomes, the animal will be born with a copy of this new information in every cell. The foreign DNA must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage.

If this does not occur, only a few cells will integrate the gene. For this reason, the DNA is introduced into the fertilized egg at the earliest stage, which is the pronuclear period immediately following fertilization. For several hours following the entry of the sperm into the oocyte, the male and the female pronuclei are visible as individuals under normal light microscope and not fused into a so-called zygote. 

The DNA may be injected into either of these pronuclei with no difference in results. Usually the injection is into the male pronucleus because it is larger than the female nucleus and also because it is closer to the oocyte surface.

These oocytes are subsequently transferred into the uterus of pseudopregnant recipient animals and develop to term.

Dolly is not transgenic

Let's be perfectly clear about this. Dolly is a clone made by somatic cell nuclear transfer. She is not transgenic.

Somatic nuclear transfer


http://www.accessexcellence.org/WN/SUA09/DOLLY.JPG

To combine our techniques of somatic cell genetics and transgenic production:

How exactly do we knock out a "gene X" in a cell line?


Now select for G418 resistance in the cell line, and add the resistant cells to an early embryo. Some of the animals in the litter will be mosaic, in that a fraction of their somatic cells will be hemizygous (have only one copy of gene X). You should identify these animals by a polymerase chain reaction test on their blood. A fraction of the animals in this group will also be mosaic in their germ lines, which you determine by testing for progeny that are purely hemizygous. Once you find such a mouse you're in business! Back-cross its progeny to make a homozygous mouse, and you've got what is called a "knockout mouse" (a mouse lacking gene X)!

Of course this is a rather crude mutation - simply obliterating the gene. There is a way of making a more sophisticated allele of gene X that contains only a point mutation. You first make a tagged mutation replacing gene X with a G418 resistance gene and HSV-TK, then use the ability to select against TK expression with ganciclovir to select for a second recombination event with a mutated allele of gene X:


Now you "simply" introduce these ES cells into an early embryo and look for mosaic mice again. This is actually fairly arduous work.

But don't be alarmed. If you're in a lab that has more money than talent, you can always hire someone else to do your work for you.