Molecular Techniques in Developmental Biology - Lecture Notes | ZOOLOGY 470, Study notes of Zoology

Download Molecular Techniques in Developmental Biology - Lecture Notes | ZOOLOGY 470 and more Study notes Zoology in PDF only on Docsity! Zoology 470 – 2009 – Course handouts Page 9 Molecular Techniques in Developmental Biology Big idea: DNA encodes RNA, which, after appropriate processing, produces a messenger RNA (mRNA). mRNAs in turn are translated into proteins. Thus by working with DNA, we are working with information required to make a protein. DNA is a very stable molecule that has convenient properties we can take advantage of. RNA, on the other hand, is highly unstable, and tends to degrade rapidly, and proteins can only be studied biochemically. Molecular biology is the powerful technology that allows us to work with DNA (and RNA and proteins), using a number of different techniques. Electrophoresis Big idea: Separate proteins or nucleic acids (DNA, RNA) by size by driving them through a gel (made of a polymer, like polyacrylamide, or a gel, like agarose). Why do this? Since DNA and RNA are made of four simple building blocks in long chains, we can’t tell them apart chemically. It’s the number of bases and their order that matters. This technique at least allows us to distinguish different DNAs or RNAs by size, if not by the sequence of the bases they contain. Campbell, 5e, Fig. 17.3 Zoology 470 – 2009 – Course handouts Page 10 Nucleic Acid Hybridization Big idea: Remember that DNA can undergo base pairing with a complementary strand of DNA, or with a complementary strand of RNA; this process is essential for replication and transcription. However, largely complementary sequences can do this, too. Furthermore, this process is reversible; heating up a solution of nucleic in the presence of low salt tends to cause complementary sequences to fall apart (“melt”); whereas lower temperatures and higher salt solutions allow them to stick together (“hybridize”, or “anneal”). Even if the nucleic acid we want to detect is immobilized in a fixed cell or on a membrane, we can still make use of this key property. In that case, the sequence we use to hybridize to the membrane or tissue is called a probe. see Purves et al., 4e, Fig. 13.1 DNA Cloning Big idea: We want to get DNA in a form that is useful for making lots of it. To do this, we chop the DNA with a restriction enzyme, producing sticky ends, and then we ligate the sticky DNA into a vector (a piece of bacterial or yeast DNA that has been similarly rended sticky). Then we let bacteria do all the work of making this new DNA for us. In a variation of this technique, we first take mRNA and make complementary, or cDNA, by using an enzyme called reverse transcriptase. Then we make this single stranded DNA double stranded, and then we clone this DNA. See Campbell, 5e, Fig. 20.2 Zoology 470 – 2009 – Course handouts Page 13 Southern and Northern Blotting Big idea: Separate DNA or RNA using gel electrophoresis, then “blot” the nucleic acid onto a membrane that’s really sticky, making a replica of the stuff in the gel. Then, probe the membrane with a labeled piece of DNA or RNA (it might be radioactive, or tagged with a molecule that allows it to be detected using a specific chemical reaction, etc.). This allows you to see if your mix of RNA or DNA contained the specific molecule that you’re looking for, since hybridization under the right conditions only allows good matches to remain annealed. When the blotted nucleic acid is DNA, it's called a Southern blot (after its inventor, Ed Southern). When it's RNA, it's called a Northern blot (to be cute; a Northern is shown here). Once a blot is probed, the blot can be placed against photographic film and exposed to reveal where the probe bound. Zoology 470 – 2009 – Course handouts Page 14 RNAse Protection Big idea: RNAse protection is used in situations when you would use a Northern blot, but is more sensitive. It uses a radiactive RNA to bind to mRNA; such pieces are protected from being digested by RNAse, an enzyme that chews up single-strands of RNA. The protected fragments are then run on a gel to separate them by size. . Gilbert, 6e, Fig. 4.14 see Kalthoff, 2e, Fig. 17.m1 Zoology 470 – 2009 – Course handouts Page 15 In Situ Hybridization Big idea: In situ hybridization is used to detect the time and location in a tissue of expression of a mRNA by allowing a complementary RNA or DNA probe to bind to the tissue. DNA Microarray Introduction: DNA microarrays (colloquially called “DNA chips”) are used to look for differences in the expression of specific genes between two tissues. A robot places spots of DNA corresponding to known genes in an ordered array on a glass slide. The array can then be probed using fluorescent DNA or RNA probes. A machine then measures the fluorescence intensity at each spot, and higher or lower intensities are measured. For example, microarrays have been used to look for differences in gene expression in cancer cells vs. normal cells, and ovaries vs. somatic tissues. See Gilbert 8e, Fig. 4.13. A related technique involves DNA "macroarrays", which contain larger spots (see Gilbert 8e, Fig. 4.14). Gilbert, 8e, Fig. 4.16 Campbell, 5e, Fig. 20.9 Zoology 470 – 2009 – Course handouts Page 18 Altering Gene Function in Embryos Introduction: Placing a molecule "at the scene of the crime" during development isn't enough to show that it actually plays a functional role in a developmental process. Ultimately, we want to perform experiments that test the role particular proteins or RNAs play in embryonic development. Here again, we make use of DNA and RNA constructs, which when combined with the cellular transcriptional and translational machinery of the cells of the embryo, result in the disruption of the function of specific gene products. Depending on the model system we are using, we use the most appropriate technique from the set listed in the following pages. Overexpression of Injected mRNA Big idea: If we inject a synthetic mRNA into a cell, the protein encoded by the RNA will be translated, and we can then assay for the subsequent effects on the developing embryo. In the example shown here, a frog oocyte is injected with a mRNA encoding a receptor protein. Many other twists on this are possible, including injecting only a few specific cells in a more advanced embryo. Antisense, RNAi, and Morpholinos Big idea: Remember that RNA can undergo base pairing with a complementary strand of nuclei acid. If the complementary piece of nucleic acid (either a short stretch of DNA or RNA) leaves a piece of a partially duplexed mRNA in a single-stranded state, then an enzyme, called RNAse H, present in the cells destroys the mRNA with the single stranded stretch. The result is the destruction of the endogenous mRNA, i.e., a depletion of that RNA. The DNA strand complementary to the strand that produce mRNA is usually called the “anti-sense” strand, hence the name of this technique. In a much more common, and related, technique, double-stranded RNA is introduced into a cell, resulting in the inactivation of the gene of interest in a process known as RNA interference (RNAi). RNAi relies on the production of small, inhibitory RNAs (siRNAs) that interfere with transcription. In some cells/embryos, short hairpin RNAs can be expressed using a promoter that drives expression in a particular tissue, resulting in production of siRNA only in those tissues expressing the hairpin. Although the details differ, RNAi works well in C. elegans and other embryos and tissue culture cells. Finally, translation of RNAs can be inhibited using chemically modified nucleic acids (morpholino oligonucleotides, or morpholinos for short). Morpholinos work well in zebrafish, frogs, sea urchins, and other species. Adapted from Kalthoff, 1e, Fig. 4.18 Purves, 6e Fig. 17.12 RNA Interference (RNAi) Double stranded RNA is introduced into a cell Double stranded RNA is chopped into pieces by an enzymeRNA Interference (RNAi) The pieces “guide” a set of proteins to the normal mRNA The normal mRNA is clipped into pieces and destroyed Courtesy of Nature journals 3' 5' translation start site mRNA for Gene X morpholino oligonucleotide against Gene X X Morpholinos Antisense RNA RNA interference (RNAi) Zoology 470 – 2009 – Course handouts Page 19 Transgenic Animals Big idea: In some organisms, it is possible to introduce foreign DNA which will be propagated in the germ line (i.e., eggs or sperm) of the maturing animals. Sometimes this results in the incorporation of the DNA into a random spot on a chromosome (“integration”); in other cases the organism can propagate the extra DNA as an extrachromosomal array, similar to a bacterial plasmid. The result is a genetically engineered animal. Note, however, that we have not removed any of the genes normally found in the animal; we have only added our foreign DNA. Such animals are called “transgenic” animals. The method used in mice is shown here. Making Targeted Mutations by Homologous Recombination Big idea: In mice (and a few simpler organisms) it is possible to replace the normal piece of DNA with an engineered piece of DNA at a particular site along a chromosome. This is done by making use of the normal recombination machinery inside the cell to produce a double cross-over event that swaps our engineered piece of DNA for the normal stretch of DNA. Often, the goal is to produce a mutations in a gene of interest that renders it non-functional. Homozygous mutant offspring are colloquially referred to as “knockout mice”. The desired recombination events are very rare, and most insertions do not occur at the right site. To select for such double recombination events, we use two different antibiotics and tricks that rely on the biology of early development in mammals. In some cases, a reporter gene is inserted at the same time as part of the recombination. Such "knock-in" alleles can be useful for detected where the endogenous gene would have been expressed. In the classic method, there are four steps to the knockout process: Step 1: Introduce the Foreign DNA into ES Cells Big idea: Embryonic stem cells, or ES cells, are cultured cells derived from the inner cell mass of mouse embryos which retain the ability to produce any type of differentiated mouse tissue. We can introduce our foreign DNA, which contains homologous sequences on its ends that match the gene of interest but have internal, engineered sequences in strategic locations. There are two important features of the engineered DNA: (1) a neomycin resistance (neor) gene that confers on cells that get it the ability to survive in the presence of the antibiotic neomycin, and (2) on the ends of the engineered fragment is DNA that encodes for the gene thymidine kinase. Mice don’t normally make this protein; cells that do die in the presence of a second antibiotic, gancyclovir. Kalthoff, 2e Fig. 15.21 Zoology 470 – 2009 – Course handouts Page 20 Step 2: Select for Homologous Recombinants Big idea: Introduced DNA can be inserted randomly or into the homologous site by recombination. Cells that have the foreign DNA incorporated at any site will be resistant to neomycin. Only if the DNA is inserted at the homologous site will the thymidine kinase sequences be excised and the cell be resistant to gancyclovir. This double drug selection selects ES cells that have our DNA inserted via homologous recombination. Step 3: Insert “Knocked Out” ES cells into the Host Embryo Big idea: Now we have ES cells that carry one “knocked out” copy of our gene, i.e., they are heterozygous for an engineered mutation in our gene of interest. We insert these cells into a host embryo. In some cases, the inserted cells will give rise to gametes carrying the engineered mutation, and the result will be that we can breed such mice to eventually obtain homozygous mutant offspring. Step 4: Breed Chimeric Mice to Get Homozygotes Big idea: The embryos that receive transplanted ES cells are actually a mixture of genetically distinct cells known as a chinera. Using mice with different coat colors, we can identify chimeric mice who may have some gametes derived from the introduced ES cells. By successively breeding these mice and their offspring, we can get homozygotes in which both copies of the gene of interest are knocked out. Sometimes the homozygotes die as embryos; in other cases they may survive but have abnormalities. The observed phenotypes are of great interest, because they tell us about the functions of our gene. Figures in this section adapted from Capecchi, 1994. Sci. Am. 270(3):52-59