Module IV Flashcards
- Describe the differences between outbred and inbred animals, and in which ways different strains can differ from one another, and how they can suffer due to their genetic constitution
Outbred colonies: they are maintained by random mating and there is a certain gene pool in the colony. In theory, there should be genetic equilibrium in the colony following the Hardy-Weinberg equation. Unintended selection will always occur if random mating occurs as some animals grow more, breed better, survive better, etc and some commercial breeders have intendedly selected those animals as they are better for economy. It may be a good idea to systematize the randomness, especially in small colonies, this can be done by rotational breeding.
Inbred animals are originally based upon outbred animals, which have then been inbred. To make inbred animals you breed close relatives, this means brother and sister or father and daughter for at least 20 generations. During inbreeding, lines will be closed down due to the appearance of recessive lethal or defect genes.
Main advantage of using outbred mice is that they are cheaper; inbred animals are far more expensive. Main disadvantage of outbred animals is that the stocks are quite different between breeders which hampers reproducibility.
- Describe appropriate breeding programs for laboratory animals
Rats and mice are fertile around the age of seven weeks, and from then on they can be mated. You can mate either one female to one male or you can let the male have several females. You will have to take into account that the female has an estrous cycle, and she will only allow mating during estrous. The morning after the mating has taken place there will be a vaginal plug of the female left by the sperm secretions from the male.
Continuous Mating - The male can either be with the females all the time.
Cycle Mating - remove the male every time when having mated and let the females stay alone in a cage with their offspring
When propagating inbred animals for research, it is too inefficient to produce all animals as a result of a brother X sister mating, and it is therefore accepted that study animals may be up to three generations away from brother X sister mating; this is called pyramidal breeding. To propagate inbred animals for research, the breeder will take a brother and a sister - the stem pair. They will get a litter, which are all brothers and sisters - The Nucleus. The brothers and sisters will be mated and produce off-spring, which will be used as breeding animals in the green production stock 1. Although these animals may be cousins they are mated randomly. They may either be used for research or it may be used as breeding animals in the yellow production stock two. So, it continues to the red production stock 3, from which all offspring can only be used for research and not for further breeding. So there is only brother X sister mating when producing the nucleus, while all research animals are the product of first, second, or third generation random mating.
- Describe how genetically altered animals can be generated and how they can be used for scientific research
Pronucleus Microinjection - The desired DNA construct is microinjected into one of the pronuclei, typically the male pronucleus, of the fertilized egg. Inside the nucleus there are always some random incidental DNA breaks, which are repaired by the restriction enzymes, and if a construct has been inserted there is a good chance that it will be incorporated into one of these breaks. The incorporation will be at a randomly selected location, that means where an incidental break occurred, and the manipulating operating person is without influence on this. Therefore, this method can only be used for inserting genes, but not for knocking out genes. As it is only done in the male pronucleus the outcome will be heterozygotic – or the term hemizygotic is preferred in mouse gene technology because there is no opposing allele, so to produce a homozygote breeding for a couple of generations is needed. On the other hand, the hemizygote will also be able to produce the gene product.
Targeted Mutation by Embryonic Stem Cell Transfection - Embryonic stem cells from the blastocysts of some very specific inbred strains of rats and mice can be harvested and grown in culture. When in culture, the whole set of gene modification techniques can be applied, which enables that after gene modification techniques specific cultivations can select only those cells in which a targeted mutation has occurred. These can be injected into another blastocyst, which is then injected into an anesthetized pseudopregnant female mouse made by mating with a sterilized male. She will give birth to chimeras, that means mice containing both wild type cells and gene modified cells. A chimeric male will be able to produce both wild type sperm and gene modified sperm. If mated with an ordinary female, there is a chance of getting both females and males with gene modified germ cells, while the pure wild type offspring can be discarded. The heterozygous male and female are mated and there is a chance that in the offspring there will be homozygotes of both wild type and gene modified types, as well as heterozygotes. Hopefully both sexes will be represented, so that it is now possible to continue breeding with homozygotic gene modified mice.
Cloning and Nuclear Transfer - Instead of embryonic stem cells from a blastocyst, one will take a cell (e.g. fibroblast) from the intact animal and cultivate it, and do the gene modification on this cell culture. The nucleus is removed from a sow’s egg, and a gene modified cell nucleus is placed instead. After further development to a blastocyst the gene modified embryo is put into the uterus of a pseudopregnant sow, who will give birth to a gene modified piglet.
Nuclease Techniques - Different nuclease based techniques, known as zink-finger, TALEN or CRISPR-cas9, have entered the gene modification laboratories to make life easier for people, who want to genetically modify animals. A nuclease is an enzyme which can induce a break in a DNA string. CRISPR stands for Clustered regularly interspaced short palindromic repeats, which are DNA sequences which have been isolated from bacteria. In the bacteria, they are linked to the nuclease cas9, which can induce a double strand break in the DNA. Basically, when it is performed on a mammal cell, this break when directed into a functional gene by the orientation of the CRISPR sequence will be enough to stop the function of this gene. This means a knockout has been created. However, if a DNA construct has been inserted this may in the repair process of the cell may be integrated where the breaks are. As such, CRISPR-cas9 can be used for targeted insertion of genes, either in a functional gene, that means a knock-in has been made, or in the non-coding region, that means an ordinary transgenic cell has been made. CRISPR-cas9 can be done in relation to a microinjection, or it can be done on embryonic stem cells in culture. It works on all animal species.
- Describe principles of genetic monitoring of laboratory animals
Genetic monitoring is performed to ensure that the genetic strain that you are using actually has the intended genotype. To do this, take a sample from the mouse and purify the DNA.
● PCR is typically used for identifying single genes; typically a transgene that we want to show the presence of it in the individual mice.
● Southern Blot is also used to identify single genes; also typically transgenes or knockouts. Normally it is used because those who produced the transgenic strain moved the cutting sites for southern blot (when inserting the nonsense region, the cutting sites have been moved). This means that when it is cut, you will get DNA pieces of different lengths and they are easy to identify because they move differently in the gel. Southern blot can also be used for the identification of strain or stocks.
● Single Nucleotide Polymorphisms (SNPs) are used to characterize strains or stocks. They test thousands of polymorphisms on all the chromosomes. They are stable over generations and therefore they can be used to characterize an inbred mouse, but it is costly compared to the other methods.
● Short Tandem Repeats (STRs) are short repeats of a double nucleotide. They are often found in non-coding regions, and they can be used to show inter-individual variation between inbred mice of the same strain. It is also a costly technique.
- Explain international standards for nomenclature of laboratory animals
In inbred animals, the line is called a strain. Whereas in outbred animals, the line is called a stock.
Substrains are designated by writing all the codes of all the relevant breeders with the historically oldest breeder first and the present breeder last.
When making hybrids, you need to first give the name of the mother strain, then the name of the father strain, then F1 for F1 hybrids, then slash for inbred and then the breeders lab code.
Some rodents are gene modified, the gene modification will be placed in a mouse strain; the background strain. So, the first part of the name is the name of the background strain, and after that will come a gene name. To separate the strain name and the gene name we write a dash (-). There are specific ways to write gene names for each species. Gene names are normally written in italics. Human genes are always written with all letters as capital letters, as for instance the human APOA1 transport protein, while the mouse or rat version of the gene would be written with the first letter as capital letter, and most of the remaining part of the name as lowercase letters.
If it is a transgene we do not write in italics, and we set brackets around the name. To designate that we have inserted a human gene as a transgene, we write Tg, and if this is done in a non-homologous way, e.g. by microinjection, we write N for non-homologous. If the mouse is a knockin, that means the transgene has been placed in a homologous way, we write H for homologous. As most inserted transgenes are non-homologous, this part of the name is often omitted. Then we write the number of the founding line. If there is only one line released, then the founder would often just call it number one, and even more often not write the number itself. As the last part of the gene name, we write the founder’s lab code, in this case Rub for Lawrence Berkeley Laboratory in California. Then we place a slash and the lab code of the breeder actually breeding the mouse; in this case J for the Jackson laboratories.
If we are naming a knockout, we write the name of the actual background strain, and after a dot, we write the donor of the stem cells, if there is such a donor. After the slash, we first write the name of the animal version of the gene, that means the gene that a normal B6 mouse would be carrying. To show that the gene function has been deleted, we write the remaining information in superscript. First we write tm for targeted mutation (tm is for classical KO mice made by ESC method, with an endonuclease mutation we use code em). Then, we write the number of the founding line, and then the founders lab code, in this case Unc for University of Northern Carolina. Finally, we write a slash and the lab code for the breeder actually breeding the mouse in the present version.
