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In this article we will discuss about the isolation of animal tissue culture.
Before culture, animal materials must be washed in BSS (balanced salt solution) aseptically to avoid contamination. The tissue to be cultured should be properly sterilised with 70% ethanol, and remove surgically under aseptic conditions. Thus the tissue isolated should be either stored in the freeze or used immediately. The following are the various stages of culturing the tissue.
Disaggregation of the Tissue:
There are two methods of disaggregating the tissue:
Physical (mechanical) and Enzymatic disaggregation.
a) Physical disaggregation:
The aseptically isolated tissue is kept in a sieve of 100µm sieve. It is put in a sterile Petri dish containing buffered medium with balanced salt solution. The cells are alternately passed through sieves of decreasing pore size (50µm and 20µm mesh).
The disaggregated cells are now counted using a haemocytometer. The viable dissociated cells are now termed as “Primary cells”. When the primary cells are grown on culture medium in high density, this culture is known as primary adherent cell culture.
b) Enzymatic disaggregation:
Enzymes are also used for dislodging the cells of tissue. By using enzymes a high number of cells are obtained. Disaggregation of embryonic tissue occurs more readily than that of adults or new borns. There are two important enzymes used in tissue disaggregation— collagenase and trypsin.
Use of collagenase:
Collagenase is used for disaggregation of embryonic, normal as well as malignant tissues. The intracellular matrix contains collage; therefore collagenase disaggregates normal and malignant tissues.
First the biopsy tissues are kept in medium containing antibiotics. Thereafter the tissue to be disaggregated is dissected into pieces in BSS containing antibiotics. The chopped tissue after washing is transferred incomplete medium containing collagenase. After five days of treatment the mixture is pipetted so that the medium may get dispersed and the epithelial cells are settled.
The settled clusters of the epithelial cells are washed thoroughly and the cell suspension is made free from the enzyme collagenase by centrifugation. The suspension consists of enriched fibroblast fraction which is plated out on medium. The cells grown are the primary cells and the culture is the primary cell culture.
Genetic engineering has enabled scientists to transfer genes across species, families, and even kingdoms. These boundaries, once thought of as impossible in the past, are now readily crossed with modern gene transfer methods that allow the transfer of DNA into living cells and fertilised eggs of animals.
Transgenic animals not only provide research tools for studying new gene regulation and disease, but they may be genetically modified for the production of pharmaceuticals, vaccines, and rare chemicals as well as for food production.
Animal Gene Transfer:
One challenge in creating transgenic animals is making sure that the transgene is turned on at the right time and in the right tissue. The integrated gene must be expressed and regulated appropriately (for example, in a tissue- specific manner). So the gene needs to be accompanied by the correct promoters and regulatory sequences such as enhancers.
Also, the gene must be in as close to the correct location in the genome as possible. The construct must then also be incorporated into the chromosome where gene expression is regulated. Two methods of introduction of genes into cells are microinjection and embryonic stem cell gene transfer.
Microinjection:
This is generally the method of choice for creating transgenic animals. Once a gene has been characterised and expressed in eukaryotic cells, a transgenic animal can be made by microinjection of the cloned gene into the fertilised egg of a donor animal.
The key here is that the foreign gene must be injected into the cell before the first cell division, so that all cells of the organism contain the desired gene. Donor females are superovulated (produce multiple eggs) and mated, and the fertilised eggs are removed and placed in a sterile dish with buffer.
DNA is injected into the eggs and is implanted into the oviduct of a female mouse made to have many of the symptoms of pregnancy by hormones. After birth, to find which of the offspring are transgenic, animals are subjected to tissue analysis by Southern blotting or PCR.
Founder animals, which are animals that have the foreign gene within their germ line (sex cells) and the somatic cells, are bred to establish new genetic lines with desired characteristics. However, there are problems because few injected eggs survive, and not all of those retain the new DNA.
The new DNA is integrated at random if it is not targeted to a specific chromosomal location. Microinjected genes often integrate into a single chromosomal location, regardless of whether the location is used a lot or not used very often. Also, many transgenic animals are mixtures (mosaics) because the gene is only in some of the cells. In order for this to be effective, all of the somatic cells, as well as the germ cells, need to have the foreign gene.
Embryonic Stem Cell Gene Transfer:
Embryonic stem (ES) cells are also used for producing transgenic animals. ES cells are obtained from the inner cell mass of donor blastocysts (fertilised eggs that have undergone several cell divisions to for a hollow ball of cells called the blastocyst).
The cells are cultured prior to transfection with a foreign gene. A gene is targeted to specific areas by homologous recombination and the use of selectable markers (antibiotic resistance). Treated cells are then screened either by PCR or another selection method such as antibiotics.
Transformed ES cells are microinjected into blastocysts so that they can become established in the somatic and germ line tissues. They are then passed on to successive generations by breeding founder animals.
At present, the ES method is most successful with mice because mouse ES cells are pluripotent (can differentiate into many cell types) and, when integrated into blastocysts, can divide and differentiate in the mouse embryo. However, today the cells do not need to be pluripotent when placed in blastocyst embryos.
The mouse is used as a model in the study of gene function, and is useful in many areas of medicine. Methods are used to inactivate gene function and look for changes in the phenotype, and gene targeting can also be used to add genes encoding desirable characteristics. ES cell transformation technology has been used to inactivate specific genes in order to determine their effect on growth and development or to develop effective treatments.
Gene Targeting:
The insertion of DNA into a specific chromosomal location, and is often used to inactivate a specific gene into the genome.
It occurs in the following steps:
1. A specific gene is isolated, altered according to the requirements of the experiment, and then targeted to its counterpart on a specific chromosome in cells. A part of the gene is inactivated by inserting an antibiotic resistance gene into it, with regions of the original gene flanking the marker remaining on either side. This marker allows cells with the gene to be selected.
This is called positive selection. A second marker (called negative selection), usually the herpes simplex virus type I thymidine kinase (HSV-tk) gene, is located at the end of the targeting vector. This marker is used to select for cells in which the transferred gene is targeted to the specific site, rather than a random site or nowhere at all.
The gene construct is assembled and the vector is placed into an embryonic host cell, where homologous recombination allows the gene to insert into a specific chromosome location.
Ideally, the target gene, antibiotic resistance gene, but NOT the thymidine kinase genes, are transferred to the chromosome. The tk gene is not transferred because it is out of the regions of the regions that we want to enter the chromosome.
2. Selection of cells: You want to select for cells with the foreign gene. Cells are placed in a medium containing antibiotic (usually neomycin), which kills cells where the gene is not inserted into the genome. Also, a second drug called gancyclovir selects for targeted insertion by killing any cells which contain the tk gene (remember that the tk gene is something that we don’t want to enter the cell).
What we want is the antibiotic resistance gene entering the cells (so the antibiotic doesn’t kill the cells), and the tk gene to NOT enter the cells (or else the gancyclovir will kill them).
If the cells have undergone homologous recombination, they will survive treatment with neomycin and gancyclovir. If we get the correct ES cells, then they are placed into mouse embryos, where they become incorporated into the genome. These genetically manipulated embryos are placed in surrogate mothers and develop into young.