Genetics Exam 3

The fungal ascus

-Meiosis well studied in this-the process takes places in a bag called an ascus and contains the products of a single meiosis- this is isolated and away from other potential meiosis events -A cross of two haploids fungi that differ at a single gene and yield as ascus containing an equal proportion of each genotype -In a crassa, you first get two cells doing dna replication then meiosis 1, meiosis 2, and finally a round of mitosis. This results in 8 cells being arranged in a linear fashion called an octad-this is because it is too thin to move past each other- no recombination and there is a 50:50 ratio of phenotypes (assuming different alleles). But one day Zickler noticed that the ratio of phenotypes was 6:2 instead of 50:50- this created the double-strand break model for homologous recombination. Zickler used the term gene conversions to describe this phenomenon- when gene conversion occurs, one allele is converted to the allele on the homologous chromosome.

Transgenic mice

-introduce genes into embryonic stem cells-these genes will integrate into chromosomes. You screen the cells for desired integration events. You then make the desired mice from these stem cells that are undifferentiated.

Two ways to repair double strand dna break (and 3 reasons why this happens)

-worst kind of damage because you can get large scale chromosome changes such as 1. Breakage of chromosome into pieces 2. Caused by ionizing radiation, free radicals, and chemical mutagens - 10-100 of these happen every day 3. Breaks cause chromosomal deficiencies and rearrangements 2 ways to repair: 1. Non homologous end joining- quick and easy (NHEJ)-somatic cells- blood, colon cells, or skin cells get replaced very quickly. This is repaired via End binding proteins-bind to either side of the broken DNA- bridging happens between these two proteins. A few nucleotides get chopped away and then it gets ligated together. The ligation removes the nucleotides. 2. Homologous combination repair

3 phases of crispr system

1. Adaptation phase: also called spacer acquisition Cas1 and cas2 protein complex cleaves invading bacteriophage dna- a piece of this dna between 20 and 50 bp is inserted into the crispr gene. The other spaces in the bacteria are due to past infections and spacing is passed to daughter cells 2. Expression phase: after adaptation, the exposure will result in the expression of the crispr, tracr, and cas9 genes 3. Interference phase: Each spacer in an cr-rna is complementary to one strand of the bacteriophage dna Cr-rna acts as a guide for the cas9, tracr, and crrrna complex to bind to that strand. Cas9 protein functions as an endonuclease that makes double stranded dna breaks- the phage proliferation is therefore inhibited.

RT-PCR a gene and clone it into a vector procedure

1. Purify the mrna from your cells of interest and anneal to it a pcr primer that anneals next to the poly-a tail. You then add reverse transcriptase and nucleotides (dNTP's)- this makes a dna copy of your mrna strand. You then do pcr. 2. Add pcr primers which copies the dna segment over and over. The segments are not that big because the mrna has no introns- this results in very small pcr fragments.

Four ways to clone a gene

1. Shotgun clone genomic DNA into a vector and identify gene of interest 2. Make a cDNA library and shotgun clone this into a vector and then identify gene of interest 3. PCR amplify the gene and clone it into a vector 4. RT-PCR a gene and clone it into a vector

Western Blot Steps

1. Total protein from the cell is mixed with negatively charged detergent called sds-this sds globs unto proteins and causes it to stretch like dna. Sds denatures the natural 3d shape of the proteins- they have to be reformed later to be detected by the antibodies. The negative charges in the sds overwhelm any positive charges on the protein 2. The sds and protein complexes are run out on a gel (usually polyacrylamide)-these kinds of gels are better for smaller purposes like protein (vs dna) 3. The gel containing the sds and protein will be transferred to a membrane (nitrocellulose or pvdf) using a western blot apparatus. An electrical current is used to transfer the protein from the gel unto the membrane. 4. The protein is then exposed to an antibody that binds to the protein of interest. This primary antibody will come from another animal like rabbit, mouse, rat, etc. 5. A second antibody that recognizes the first antibody and furthermore has a chemiluscent molecule or reporter is attached to it-this is usually HRP. You then add a substrate that reacts with the reporter- to produce the light. 6. The membrane is then exposed to a photographic film- the black bands on the film represent the protein of interest

Using crispr to make null alleles:

1.Add cas9 and rna unto the gene body (but after the promoter) 2.The dna is cleaved and NHEJ repairs the break- a few bases get removed 3. Result is a small deletion mutation-creates a frameshift in ⅔ of repaired chromosomes

Knock-in Mice

A gene addition in which the gene of interest has been added to a noncritical site in the genome- would have to be in the euchromatin and would be added via homologous recombination- no other genes are changed or added.

Why do we use gene knockouts on mice:

A strain of mice engineered to carry a mutation analogous to a disease causing mutation in a human gene is termed a mouse model Gene knockouts often reveal the function of a gene because one can study the mutant mice more the human patients Sometimes there is no phenotype due to gene redundancy- this is about ⅓ of the time- they may also have a phenotype that we cannot detect

Using crispr to insert dna

Add cas9 and rna- attached to the gene body- you cleave the dna (double strand) and insert the dna (gene and its promoter) which is single stranded. Half of the sequence is the host sequence and half is the new sequence. This results in homology directed repair which uses ultramer to ligate the dna into place People have also added crispr to a fertilized egg-there is about one day before the nuclei fuse together-we add cas9 and rna at this point-this modifies the gene and creates a mutant offspring. If a gene was disrupted by a frameshift mutation, then 75-85% of the mice will have the mutation.

RT-PCR a gene and clone it into a vector advantages vs disadvantages

Advantages: Fast and easy Disadvantages: Does not have a promoter (you could do regular pcr and get the promoter tho) Cdna does not work well as a transgene Pcr is error prone- carefully not to introduce mutations Rna can produce many splice products resulting in multiple proteins

Antibodies

Antibodies- proteins made by the immune system to recognize foreign substances like viruses or bacteria and target them for destruction- the foreign substances are called antigens. Antigen and antibody recognition is very specific-each antibody recognizes a single epitope. Each b cell, a type of white blood cell, produces a single type of antibody which sits on the membrane of the cell. In addition, each B cell produces many daughter cells that turn into plasma cells which secrete many copies of its antibodies into the bloodstream.

Antibody light chain locus

Antibody light chain: the genes for these are composed of various sequences or domains. 300 domains are known as variable V, four domains known as joining J, and one domain known as constant C. this has to do with dna splicing. There are 1200 possible combinations for the light chain. Immunoglobulin light chain locus: 1. There are 300 V domains followed by 4 joining domains followed by 1 constant domain. First Rag1 and Rag2 recognize recombination signal sequences and breakdown the molecule at a random V domain and a random J domain (different in every cell). The dna sequence between these two domains is lost. 2. Then, NHEJ proteins catalyze the joining of v domain and j domain-this is called nonhomologous dna end joining. This fusion process is not very precise so some bases may be lost- this is a very messy process and creates diversity in antibodies. The gene is then transcribed into a pre-mrna starting at the last variable domain because there is a promoter at this region 3. The region between the first joining domain and the constant domain is spliced out in the pre mrna. The mrna is then translated into a polypeptide and contains one variable, one joining, and one constant.

chromosomal vs vector dna

Chromosomal dna= original source of dna segment of interest Vector dna= serves as the carrier of the dna segment that needs to be cloned

The recombination process can cause the gene conversion to occur in one of two ways

DNA mismatch repair Process: when you have a crossover, it migrates down the chromosome-when it migrates there might be a few sites where the dna is not identical- this create mismatches and dna mismatch repair will randomly fix that and remove one of the nucleotides. Dna gap repair synthesis Process: when you make a double strand break and create a gap you are degrading one of the alleles away-the template strand must be used to fill in the gaps. The intertwined strands are then resolved.

Shotgun clone dna into a vector and identify genes of interest disadvantages vs advantages

Disadvantages Gene may be too big for a plasmid- usually up to 20 kB- you have to put them in BACS which can handle 300K in size More difficult to clone large dna fragments Advantages Have promoter Gene in genomic dna is usually expressed better if you make a transgene from it- you have your introns with you- not sure why this works better

Pcr amplify the gene and clone it into a vector advantages vs disadvantages

Disadvantages Too many genes and too large making this impractical Pcr is error prone so we need to be careful not to make too many mutations- you need very specific dna polymerases for this to be accurate Advantages Fast- not dependent on restriction enzyme cutting sites on either side of the gene Can also pcr amplify the promoter

Make a cDNA library and shotgun clone this into a vector and then identify the gene of interest advantages and disadvantages

Disadvantages: Does not have a promoter Cdna often does not get expressed or not expressed well when a transgene is made from it Genes may produce multiple rna splice products resulting in multiple proteins Advantages: Intronless gene is smaller and user friendly

B Cell antibody proliferation and differentiation

Each B cell has its antibody that it produces planted in the cell membrane with the sticky surface facing outward. When it binds to something, there is a signal produced inside the cell which tells it to proliferate. During proliferation, the variable regions of the antibody genes are deliberately mutated. The goal of this is to develop a modified antibody that binds to the antigen with higher affinity. Usually it does not get better binding, but usually worse. Those cells undergo apoptosis. Cells with better affinity survive and go on to form two kinds of cells called memory B cells or plasma membranes- both of these can find the antigen and bind to it- we just have two mechanisms for finding and destroying them. Memory cells-antibodies in cell membranes as receptors. Plasma cells- secrete antibody into blood

transcription-repair coupling factors + Procedure

In eukaryotes, several proteins have been shown to act as transcription-repair coupling factors: 1. In cockayne syndrome, two genes CSA and CSB encodes protein that have transcription-repair coupling factors 2. Some of these have been identified in people with high rates of mutations 3. In e coli, a protein known as transcription-repair coupling factor (TRCF) targets the ner system to actively transcribing genes having damaged dna Procedure: 1. Rna polymerase stops because it cannot transcribe a thymine dimer- a helicase gets recruited to this site called TRCF- this removes rna polymerase from the damaged region 2. TRCF contains a binding site for UvrA and recruits the UvrA and UvrB complex to the damaged region. TRCF is then released. NER then takes place. Overall, you have more aggressive recruitment for transcribed genes compared to non-transcribed.

Examples of medical agents made by recombinant microorganisms

Insulin- hormone that promotes glucose uptake-for diabetic patients Tissue plasminogen activator-dissolves blood clots-for heart attack victims or other arterial occlusions Superoxide dismutase-antioxidant-for heart attack victims to minimize tissue damage Factor VIII-blood clotting factor- for hemophilia patients Renin inhibitor-lowers blood pressure-for hypertension Erthropoeitin - stimulates the release of red blood cells- for anemia

CRISPR-CAS system

Many prokaryotes (bacteria) have a system called the crispr cas system that defends against foreign invaders-such as plasmids, bacteriophages, and transposons- ncrna played a key role This is a very convenient way to guide rna and includes the genomic sequences that you wish to cleave- in the kind of crispr made by companies cas9 and the guider protein are the same molecule. The cas9 then makes the double stranded breaks next to the genomic target sequence Crispr cas is an operon with 5 genes tracr, cas9, cas1, cas2, and crispr. Crispr gene has many clustered regularly interspaced short palindromic repeats- the repeat are interspersed with short unique sequences which are called spacers. The spacers are always the same length.

Microorganisms common uses:

Medicine production-antibiotics- synthesis of human insulin in e coli Food fermentation-cheese, vinegar, beer, wine Biological control-control of plant diseases, insect pests, and weeds. Symbiotic nitrogen fixation, and prevention of frost formation. Bioremediation-cleanup of environmental pollutants such as petroleum hydrocarbons recalcitrant synthetics

Pcr amplify the gene and clone it into a vector procedure

Pcr- you take genomic DNA, take primers which under heat anneal to opposite strands of the dna and the 3' ends are facing each other. Then more copies of this dna sequence get made. The end result is that you get double stranded DNA with primers as their ends (there are forward and reverse primers). The primers are right before restriction enzyme cleavage sites- you get pcr fragments with sticky ends which allows them to be cloned into the vector. THE WHITE COLONIES END UP BEING WHAT YOU ARE LOOKING FOR Note: run about 35 cycles on the thermocycler

Southern blot procedure -see if cloned copy matches the endogenous gene copy

Procedure: 1. Total genomic DNA is purified and cleaved using a restriction enzyme (you would do this to compare cancer vs normal cells for example). Total genomic dna fragments are run out on an agarose gel. The gel is then placed on top of an elevated filter paper whose sides dip down into a fluid. A nylon membrane is then placed on top of the gel. Filter papers and paper towels are placed on top of the membrane followed by a weight. The fluid moves slowly upwards into the paper towels-this carries DNA with it. This transfers DNA from the gel to the membrane. The dna cannot go past the membrane because it is positively charged. 2. The membrane is then exposed to UV light which causes the dna to be covalently linked to the membrane-the membrane is then placed in a liquid and denatured- a probe is then added to the liquid- this is single stranded dna that is 25 bases or longer. This probe is unique and becomes sealed to the protein of interest. Excess probes are washed away and exposed to an x-ray film.

Insulin Production

Production process in insulin- insulin is made up of two genes-insulin a chain gene and b chain gene They fused insulin-both chains independently to the lacz gene. They purified the fusion proteins, and then CNBr to cleave the b-galactosidase from the insulin at methionine. They then mixed the two chains together- with small proteins it is easier to renature them into the 3d state. The two chains then refold and oxidize cysteines which create disulfide bonds and make active insulin.

OG mechanism for homologous recombination-WRONG

Robin Holliday in 1964 proposed a mechanism for homologous recombination- he said that it started with a nick in single stranded dna- this was false- it started with a double stranded break-this was discovered by Szotack, Orr-Weaver, Rothstein, and Stahl. True Process: 1. A double stranded break occurs in a chromosome with a homologous chromosome- strand degradation occurs at the double stranded break site to yield single stranded ends. Strand invasion of the template strand causes D loop formation. 2. There are two ways to resolve the double crossover- the d loop has to break in one of two ways- one mechanism creates recombination and the other does not

Making a conditional knockout mice (examples)

Some genes are too important to knock out-the mice may not even be born or the cells may die. The previous method is usually used for Braca1 and braca2- these breast cancer genes are deleted in over 90% of familial breast cancer cases (heterozygous for one null allele and one normal allele)- but if you knock them out the mice cannot be born- die during embryonic development. We had to develop a gene where we knockout breast tissue only.

Bacteria vs Yeast Host Cell

The cell that harbors the vector is called the host cell Host is usually non-pathogenic strain of e coli The host cells lack the antibiotic resistance that the plasmids have so the bacterium only survives antibiotic if a plasmid is present Yeast Host Cell: Yeast has a tendency to rearrange after the cloned dna Antibiotics cannot be used to retain plasmids in yeast-instead people have an essential gene exclusively on the plasmid

somatostatin protein synthesis

The first somatostatin protein was produced in e coli-the fusion protein is made from a fusion gene. This did not work- the bacteria made the protein and then degraded it unless it was linked to bacterial protein. Art riggs a famous scientist decided to fuse somatostatin to the lacz gene- it is then linked to bacterial protein. This allows it to not get degraded. The process for this includes purifying the fusion protein, cleaving it at the junction at methionine-cyanogen bromide is used to cleave proteins that are next to methionine. This separates the somatostatin from the bacterial protein.

Immunoglobulin heavy chain polypeptides

This is very similar to the light chain but more complicated- there are 500 V segments- one of the 500 is chosen at random, 12 diversity segments, 4 joining segments, and then 1 constant which is longer than the light chain. The heavy chain in the variable, diversity, and joining region create the sticky surface in the light and heavy chain and thats what would bind to an antigen. There are 24,000 combinations for the heavy chain. One diversity region gets spliced to one of the joining segments. Then you have one of the 500 V segments spliced unto the diversity and joining segments. Then the gene gets transcribed into rna and the rna gets spliced which includes removing the gaps between the constants, getting rid of the rest of the joining domains, and getting rid of introns.

Reproductive cloning and process

This refers to the methods that produce 2 or more genetically identical animals- identical twins come from the same fertilized egg Cloning is an easier undertaking in plants- plants can be cloned from somatic cells For several decades scientists believed that mammalian somatic cells were unsuitable for cloning-in 1997 ian wilmut at the roslin institute cloned a sheep to produce dolly Protocol for cloning sheep: Take a mammary cell from sheep and an unfertilized egg without a nucleus- these were fused together. The mammary cell took over because it has so much more cytoplasm- the nucleus gets remodeled- you can then out these cells into the sheep and a cloned animal would be born

2 non-coding rnas in crispr

Two non-coding rnas: 1.Pre-crna 2. Tracr-rna- a region of this rna is complementary to the repeat sequences of the pre-crna. This results in several base pairs pairing with the bases in pre-crrna. The pre-crrna is then cleaved into many small rnas now called crRNA. The tracr-rna and crrna complex now binds cas9 protein through recognition site in tracr-rna

Vector design

antibiotic resistance gene Origin of replication Place to insert dna- the investigator inserts it there Lacz gene-encodes a protein called beta-galactosidase that can cleave a chemical called Xgal-the resulting chemical is blue after cleavage which produces blue bacteria (the inact is colorless)

RT-PCR-reverse transcriptase PCR

basically replaced northern blots- this is used to detect and quantitate the amount of rna in a living cell. This is super sensitive and can detect small amounts of rna in a single cell. Procedure: 1. Rna is isolated from a sample 2. Rna is mixed with reverse transcriptase and a primer (forward and backward-MUST HAVE 2) that will anneal to the 3' end of the rna of interest. 3. This makes a single stranded cdna which can be used as a template dna for pcr

Bioremediation or biotransformation and two types

clean up pollutants using microorganisms- they change the structure of the pollutants through the use of enzymes from the microorganism. Two types: 1. Biotransformation with biodegradation- the toxic metabolite is degraded into nontoxic metabolites 2. Biotransformation without degradation-the pollutant is rendered less toxic by oxidation or reduction reactions or polymerization reactions.

Homologous recombination

crossing over occurs frequently during meiosis 1 and sometimes during mitosis- this involves the exchange of dna between non sister chromatids between homologous chromosomes. This creates four haploid cells with recombinant dna also called non parental genotype. Crossing over that occurs between sister chromatids is called sister chromatid exchange- these are usually genetically identical to each other and thus does not produce new allele combinations. This is not usually considered recombination.

Bacteria Plasmid

extra chromosomal circles that are not essential for the bacteria. High copy number dna plasmids can yield more dna when it is purified-20 kB can be inserted. Low copy number dna plasmids can handle larger dna fragments being inserted when a bacterial chromosome of origin is used-100-200 kB can be inserted.

Virus Plasmid

have sequences removed or have mutated so that it is no longer a pathogen- dna can be inserted into them

Other ways to make a transgenic plants

main barrier is to get dna past the cell walls which are barriers: 1. Dna guns-you shoot the walls with a bullet that goes into the plant cell 2. Remove cell wall via digestion and get DNA inside using electrical current- treat it with enzymes that digest the cell wall and then mix the DNA (protoplasts) with the cells and then the cell grows back its cell wall.

Recombinant microorganisms definition

one whose DNA has been altered in the lab. Each year new strains of recombinant microorganisms are tested for their use of biological control of plants. Lots of people oppose the release of recombinant microorganisms.

Gene cloning

procedure of isolating and making many copies of a gene in the dna. One typically clones dna in a vector- it is usually a plasmid but could be a virus. We grow these plasmids in e coli (in bacteria) or yeast (less common- strong homologous recombination that can modify the plasmid dna).

Two mechanisms for Biological control

refers to the microorganisms or their products to alleviate disease or damage from environmental conditions. Two ways to do this: 1 Nonpathogens-this competes with the pathogen for nutrients or space 2. Micro-organisms that produce helpful toxins-they inhibit other insects or microorganisms but not the human or plant itself

Repair of actively transcribed dna

repaired more thoroughly than non-transcribed dna in both eukaryotes and prokaryotes The targeting of dna repair enzymes to actively transcribing genes has several advantages: 1. Active genes are more loosely packed and may be more vulnerable to dna damage 2. Transcription makes dna more vulnerable to damage 3. Dna regions with active genes make them more likely to be crucial for survival

Genetically modified plants (how to make them)

selective breeding has been used for centuries to produce plants with desirable characteristics. Genetic engineering has been used for crops since the 1990's and about 26% of all plants are transgenic. The most popular way for this is using the pathogen agrobacterium tumefaciens. When there is a plant or tree with damage but the cells are not dead, the agrobacterium can go into the cells and infect them. During infection, the T dna within the plasmid is transfered to the plant cell. The t dna becomes integrated into the plant cells dna genes within the t dna promote uncontrolled plant cell growth- what you get is called a crown gall tumor which is cancerous. So what people did it they modified the transposon- they removed the onc gene- you insert your gene of interest into the transposon- the plasmid is put into the bacteria- the transposon goes into the plant chromosome and the gene of interest is expressed.

Northern Blot

separates rna which is then run out unto a gel and probed-use it to see if your gene of interest is expressed and if it is, at what level Purify rna from the cell and run it out unto a gel- transferred to a filter and basically the same procedure for a southern with a few different chemicals.

Red Blood Cells Stem Cell Pathway

the stem cell that red blood cells come from are multipotent because they can produce multiple types of cells such as basophils, etc-this is called the myeloid progenitor and the pluripotent that makes that is named hematopoietic progenitor. The hematopoietic progenitor can also make lymphoid progenitor as well. This cell type originates from hematopoietic stem cells

Site specific recombination (procedure and viral example)

two dna sequences with little or no homology align themselves at specific sequences-these are very short and are for recombination. The breakage and reunion of the chromosomes is catalyzed by special enzymes. The following use site specific recombination: 1.Specific viruses during replication 2.Certain viruses and transposons to insert their dna into host dna 3. The mammalian immune system to create a diverse array of antibodies Replication of some viruses (phage p1) that infect bacteria that use site-specific recombinase. This virus has a circular genome- it makes a nick in its one strand of its circle (dna polymerase peels it off) and synthesizes dna using the intact circle as a template (does lagging strand synthesis). This creates linear DNA which has many double-stranded copies of the same genome and they all need to be converted into a bunch of different circles. This conversion is done by the site specific recombination- it uses loxP. The enzyme that does this is called cre recombinase which does the homologous recombination between loxp sites. After the undergo recombination, the circle is able to break off into its own genome. Every genome has its own loxP site. Finally, these genomes get copied in capsids and they can go infect other bacteria. Humans do site-specific recombination for the immune system.

Plasmids

we select for them based on antibiotic resistance to the host cell-this is one of many selectable markers that we use to select a plasmid. When antibiotics are added to the environment, only these cells can survive. The three most common antibiotic resistant plasmids that we use are: Ampicillin resistance- Ampr Chloramphenicol resistance- canr Kanamycin Resistance-KanR

Herbicide glyphosate

this kills plants and weeds- if you insert a resistance gene into the plants for herbicide then you can kill the weeds and not the plants when spraying this.

Process of making transgenic or knockout mice

1. Clone a mouse gene into a plasmid vector- 2. Remove enough of the mouse gene (not entirely) that the mutation would make the null allele and insert the neoR selectable marker 3. Insert the herpes simplex TK gene. 4. Linearize the plasmid using restriction enzymes, purify it, and transfect it into embryonic stem cells 5. 90% of the time the dna goes into a dna break in the chromosome using NHEJ- the NeoR gene goes in the break. These cells survive exposure to G418 because the neroR gene detoxifies G418. These cells die when exposed to ganciclovir because the HSV TK gene converts the nontoxic ganciclovir into a toxic substance 10% of the time the dna inserts into a chromosome via homologous recombination. These cells survive exposure to G418 because the neroR gene detoxifies G418. These cells also survive exposure to ganciclovir because the HSV TK gene was not inserted into the chromosome (and the dna that was not inserted is lost)- you can select for this cell type by using g418 or ganciclovir 6. Inject the embryonic stem cells into the blastocyst 7. Inject blastocytes into uterus of pregnant female 8. Chimeras are born- the embryos were from albino mice and the stem cells are from agouti mice- this means they are part agouti and part albino 9. NOW- you need to mate the albino with the chimera- this will tell us if they produce any sperm or eggs-you will discard any albino mice (because the gamete cells came from the host blastocyst and not the injected embryonic stem cells). The agouti offspring will be from the injected embryonic stem cells-you need to test for the mutation using pcr to be sure.

Process of making conditional knockouts

1. Clone dna from upstream and downstream of mouse gene into plasmid vectors 2. Insert NeoR and PuroR genes flanked by LoxP sites into vectors 3. Insert the Neo and Puro genes into the chromosome via homologous recombination 4. Expose cells to cre recombinase to delete NeoR and PuroR to get a floxed mouse gene (otherwise the genes may affect the mouse gene of interest expression)- one lox element before and after the gene (two lox sites get deleted)-the result is the mouse gene is uninterrupted. Only in one cell or tissue type will cre recombinase be expressed and result in that gene being deleted. 5. Then, mate the mouse to a mouse that expresses cre recombinase-from a promoter that is expressed only from the tissue of interest and mate again to get mice that are homozygous for the floxed allele. THIS MOUSE SHOULD HAVE THE FLOXED ALLELE IN ALL TISSUES BUT IN THE TISSUE OF INTEREST, IT WILL BE NULL ALLELE BECAUSE OF CRE RECOMBINING WITH THE FLOX SITES Another example of this is p56- this is highly expressed in the thymus and largely in the t cells-this can be knockout in t cells only via the conditional knockout method

DNA Mismatch repair in bacteria (3 bacteria types needed and procedure)

3 proteins in bacteria MuS- recognizes the mismatch dna and binds to it MuL-binds to MuS-acts as a linker between the other two proteins MuH- binds to MuL and scans the DNA until it encounters a hemimethylated DNA Procedure: 1. The first thing that has to be done is look at dna methylation to determine what base is correct and what is wrong - the correct base will be methylated because it will have been a part of the template strand which should already be methylated but the daughter strand will not be methylated yet 2. Mus binds, then mul, and finally muh 3. MuH makes the cut on the non methylated strand. MutU separates the dna strands at the cleavage site and an exonuclease digests the the non methylated strand and dna polymerase comes in and fills it in with dna ligase finishing the job by sealing the ends We DO NOT KNOW HOW THIS WORKS IN EUKARYOTES- humans do not have MuH- this is because we do not use DNA methylation to distinguish between the newly synthesized strands and the template strand. Eukaryotes also do not have MuS or MuL

Gene knockout collections

A broad goal is to determine all of the genes in every species genome One approach to this is to generate a collection of organisms from one species, each of which has one gene knocked out (inactive function). The new phenotype could indicate function or could combine knockouts to study pathways. Ways to generate a collection of knockouts include transposable element jumps or homologous recombination Knockout collections exist for e coli, c elegans, s cerevisiae, and mice is currently in progress

Aging of cloned animals

Evidence showed that dolly may have been genetically older than her age- at 3 years old the length of the telomeres in her somatic cells were consistent in a sheep that are 9-10 years old The sheep that donated cells to produce dolly was 6 years old-thus dolly's shorter telomeres were likely a result of the shortening of the telomeres in the donor sheep Studies in cloned mice showed 4% of all genes were not normally expressed

How to prepare chromosomal dna for cloning

First get the source of the gene- tissue, blood draw, cell line- from the organism of interest. Then, break open all the cells, purify the dna, and obtain the dna fragment of interest.

Nucleotide excision repair (NER):

General process for dna repair including thymine dimers and chemically modified bases, missing bases, and some types of cross-linking (covalent bonds between dna strands or proteins) NER in Human disease: Several human disease have been proven to be involved with defects in genes with ner including xeroderma pigmentosum and cockayne syndrome and both illness include increased sensitivity to sunlight. Procedure: -In E Coli 4 proteins are needed (found when studying dna exposure to UV light so they are called Uvr proteins or ultraviolet light repair) These repair pyrimidine dimers and recognize and remove a short sequence of damaged dna, then dna polymerase and ligase come in and finish 1. The UvrA/UvrB complex travels along the dna until it recognizes a thymine dimer and after the damage is detected, UvrA is released and this recruits UvrC which binds to the dimer and then cuts the strand with the dimer. It cuts 4-5 nucleotides on one side and 8 on the other of the dimer. UvrD which is a helicase gets rid of the cut dna (helicase always gets rid of the cut dna) and uvrc and uvrb are also released.

List of proteins that can produced in domestic animals

Lactoferrin-hosted in cattle-used in infant formula as an iron supplement TPA-goat host- dissolves blood clots Antibodies-hosted in cattle-combats infectious diseases A-1-antitrypsin-hosted in sheep-treats emphysema Factor IX-shep host-treatment of hemophilia Insulin-like growth factor-hosted in cattle- diabetes treatment

Make a cDNA library and shotgun clone this into a vector and then identify the gene of interest procedure

Procedure:purify the entire mrna and then reverse transcribed into cdna- you then clone that library into a vector and isolate gene of interested 1. Get Mrna- this has a poly A tail-add a polydT primer which anneals to the tails. Then you add a reverse transcriptase which synthesizes the cdna. Finally, rnase H will chop up and remove most of the rna. The remaining RNA will act as primers and will be used to create a double stranded cdna. 2. CDNA is then ligated to linkers which are sequences with a restriction enzyme sequence in them- this means all of your DNA fragments have sticky ends. These go into the plasmid. Note: it is easier to insert cdna into a vector if double stranded oligonucleotides containing a restriction enzyme cleavage sequence are first ligated onto the cdnas and then are cleaved to produce sticky ends. Getting the cdna into the plasmid is easier than the first method- this is because it is smaller due to the introns being gone. 3. You combine the cdna with the linker sequences with the plasmid dna and the sticky ends base pair and then dna ligase ligates them together. (has all the other same anatomy) 4. Then all other filters, etc steps

How do you make sure your cloned dna is the correct segment of dna?

Restriction digestion analysis-cleave clone dna with restriction enzymes-see if the size matches the correct fragment Procedure: Purify your plasmids with the cloned dna and aliquot them into multiple tubes- in each tube add a different restriction enzymes-some examples include BamHI, Pstl, and EcoRI. You can also mix different enzymes together to cleave many fragments of dna. You should check both the number of fragments and size when you use restriction enzymes. You then run these fragments out onto a gel via gel electrophoresis and compare the fragments to your size markers or ladder. The gel is visualized using ethidium bromide (slides into the dna between the nucleotides)- you can see it when you shine a light on them and they glow.

Transgenic sheep with human protein in milk

Sometimes the protein does not fold properly or does not get post-translationally modified like we want- this happens in bacteria. If you want to express a human gene, it will work better in another mammal like a sheep. One example is humans inject a b lactoglobulin promoter into a sheep oocyte, then implant the fertilized oocyte into a female sheep who gives birth to a transgenic sheep offspring. BEFORE THIS YOU HAVE TO GET THE GENE INTO A PLASMID IN ORDER TO INJECT IT. This promoter releases human protein in animal milk. Sometimes the gene is always expressed, sometimes it is expressed for awhile and then not, and sometimes it is never expressed.

stem cells + types

Stem cells- stem cells can form any cell type- they supply the cells that construct our body from a fertilized egg. In adults these also replace damaged cells. Stem cells are rare in adults and 1/110,000 cells are stem cells in the bone marrow. Embryonic stem cells and embryonic germ cells can be grown in the laboratory-great potential for transplant therapy. In the 1980's, smithies, evans, cappechi won the nobel prize for developing methods to engineer mutations into embryonic stem cells to derive mutant mice. These are very useful for two reasons: 1. Help us understand the basic genetic mechanisms that underlie the process of development 2. Offer the potential to treat human disease or injuries that cause cell and tissue damage (for example bone marrow transplants to treat cancer patients) Types of stem cells: Totipotent- stem cells that can make a whole organism- can make up to quadruple identical cells. This is like fertilized eggs. Pluripotent-cells can differentiate into almost all cells but these by themselves cannot become an entire individual. this is because they do not have enough cytoplasm. There are two types: embryonic stem cells and embryonic germ cells (become gametes). t Multipotent-cells can differentiate into several cell types Unipotent-cells can only differentiate into one cell type

Induced pluripotent stem cells (how created, 4 genes,

Type of pluripotent stem cells that can be generated directly from adult cells Was done by the shinya yamanaka lab in japan-in 2006 it showed that the introduction of 4 specific genes encoding transcription factors could convert adult cells into pluripotent stem cells. They did this by identifying 24 genes that were identified as being important in ESC and found that 4 of them, oct, Sox2, cmyc, and Klf4 were good enough to create embryonic stem cells each independently. They used retroviruses to deliver these genes to mouse fibroblasts to prove this. Won 2012 nobel prize for discovering that adult cells were reprogrammable UNDIFFERENTIATED CELLS DERIVED FROM DIFFERENTIATED CELLS Ips cells can propagate indefinitely and give rise to all other cell types in the body-they represent a single source of cells that can be used to replace those lost to disease or damage. This unlimited supply of cells could be used to generate transplants without the risk of immune rejection. Ips technology has not been deemed safe for patient use but they are being used to discover and research personalize drug discovery and patient specific basis of disease. Before, the generation of embryonic stem cells involved the destruction of preimplantation stage embryos and this had a lot of controversy. Because embryonic stem cells come from embryos, until this point it had been impossible to create patient matched embryonic stem cell lines (you can take a patient specific cells and add their missing or broken gene into their own cells and then reinsert them).

Making a gene replacement mouse process

You can replace mice genes with human genes Process: Insert the NeoR and gene of interest into the chromosome via homologous recombination Expose the cells to cre recombinase which gets rid of NeoR

Restriction enzymes (or restriction endonucleases)

get the target sequence of interest. These are used by the bacteria as a first line of defense against viruses. Many bacteria methylate their own dna at specific sites-they also express restriction enzymes that would cleave the dna if it were unmethylated. Viruses that infect the bacteria have unmethylated DNA and therefore can get cleaved. Restriction enzymes are usually purified and will cleave the DNA at 4 or 6 bases sequences which causes them to separate-at two defined locations with one at each end- this is common at sequences that are palindromes. Some enzymes produce blunt ends (clean break with no overhangs) and some produce sticky ends (have oberhand and hydrogen bond- easier to anneal together) . BANH is the most famous restriction enzyme. We mix the two types of sticky ends together and they ligate together using ligase- creates hybrid of vector and dna sequence

Transgenic animals (issues with this)

inject genes into fertilized eggs-the genes will integrate into the chromosomes-obtain several independently derived transgenic animals. Select out the ones that express the genes in a way that is close to what is desired. The problem is that a lot of the genome is repressive chromatin and they are silenced- you need to hope the gene is in heterochromatin and can be expressed.

Homologous combination repair (process)

slow and precise repair (HRR)-stem cells which replace old or dead cells- it is more important that these are accurate. 1. The cell takes advantage of the fact that there are two homologous chromosomes-homology sensing proteins line up the intact and broken copies of the chromosome(the sister chromatids). Proteins then come and remove some nucleotides- it goes from a break to being a gap. 2. Gap repair synthesis then happens- the intact strand acts as a template for the synthesis of dna to fill in the gaps to the chromosome with the gaps. At the end of the synthesis, you can have the two fully intact chromosomes pull apart or rip apart in the center 3. Finally, the ligation takes place If there are allelic differences between the two chromosomes in the sequence where the gap was made, then the repaired chromosomes will now code for those alleles. HRR is a subtype of homology directed repair or HDR. There are other forms of HDR that only require single strands of DNA as templates. In a double stranded break, you can add single-stranded dna into the cell and uses it as a template to fix the chromosome-

Gene therapy to correct ADA (adenosine deaminase deficiency)-

this causes an illness called SCID or severe combined immune deficiency. This therapy makes the gene in a body that does not make it correctly. They did this by removing ada lymphocytes from the scid patient in the lab and cultured the cells in the laboratory. They then infected the cells with a retrovirus that contains the normal ada gene. They then reinfuse the ada gene corrected lymphocytes back into the patient. If we had just injected dna into the cell, then the dna would get ligated together and the entire gene would get put into the chromosome somewhere and ends up in heterochromatin- viral vectors only insert one copy somewhere into the genome. They found that patients were getting leukemia because of this- this is because the retrovirus inserted near an oncogene which then got overexpressed.

Real Time Taqman

this is what is used to quantify the dna or rna- it is basically an oligonucleotide with a fluorescent reporter molecule and a quencher molecule at the other end. Due to their proximity, the quencher molecule blocks the fluorescence of the reporter molecule on the oligonucleotide. During primer extension, taq polymerase 5'-3' exonuclease activity digests the detector oligonucleotide which separates the reporter and the quencher. This makes a fluorescent signal-this takes multiple cycles for this to work- the strength of the signal is proportional to the amount of rna or dna in the sample and becomes stronger each cycle. The graph of the reaction will fall as the reactants become limiting and plateaus when one or more are completely used up. The cycle threshold or c1 depends on the initial concentration of dna. With low amounts of dna, we need more cycles to detect the fluorescence and with more dna we need less cycles to detect the dna. The initial concentration of a product can be found by comparing it with two known concentration standards. Each extension from the forward primer releases one fluorescent reporter which can be detected in the thermocycler

Base mismatch

this is when the wrong nucleotide is attached to the new dna strand- this base mismatch does not obey the AT and GC pair binding rules and may cause a tautomeric shift in the dna template strand-DNA polymerase detects this type of mutation via its proofreading ability-this wrong nucleotide slides into the exonuclease site on the new strand- the exonuclease site removes a few nucleotides and then re adds them to the strand IF PROOFREADING FAILS then the DNA mismatch repair comes to the rescue-these are found in all species and are associated with cancer in humans.

Shotgun clone dna into a vector and identify genes of interest procedure

this process involves collecting cells and growing them, isolate dna using restriction enzymes, mixing the dna with the vector (both have been cleaved) and they get covalently linked. Then the bacteria and vector get mixed together- the bacteria is treated with cells that make it permeable to the plasmids. You then incubate this overnight using media with allolactose mimic called IPTG, X-gal, and ampicillin. The bacteria grows and divides in this media. If you have x-gal in the media you will have blue bacteria and colorless- the blue are no good because they have the lacZ gene intact and have no dna. The colorless have dna inserted into them and are used. Then, a nylon filter plate is used to collect the bacteria. The filter is treated with detergent to permeabilize DNA or burst it open. The DNA is then exposed to ultraviolet light which cross-links the DNA to the membrane. NaOH is used to denature the DNA (separates the strands) and then a probe is added that is complementary to the target gene. The probe will have a sequence of the dna you are interested in-it anneals to the separated strands-and it can be radioactively tagged or fluorescently tagged. After annealing the probes, YOU MUST WASH AWAY THE UNANNEALED PROBE. You then place it next to an x ray film and the colonies containing the target gene are identified on the master plate.

Insulin

this regulates the uptake of glucose into fat and muscle cells-it is produced by beta cells in the pancreas. Persons with insulin dependent diabetes cannot synthesize enough insulin. Old sources of insulin induced cows and human cadavers- VERY EXPENSIVE AND COULD DEVELOP AN ALLERGY.

DNA Sequencing + procedure

this was invented in the 70's by a scientists named fred sanger. You use synthetic nucleotides called dideoxyribonucleotides- they are missing the oxygens on both the 2 and 3 carbon on the deoxyribose sugar group- during dna replication these create a dead end- you cannot attach the next nucleotide to the 3' carbon without the oxygen- this is called chain termination. Procedure: 1.Clone dna into a plasmid 2. primer to sequence in the plasmid 3. Sequence the dna and do four different reactions- each with a different ddNTP (one with c, g,a, and t). These synthetic oligonucleotides would be radioactive (modern is not fluorescent) so they could be imaged and visualized- these sequences were then run out on a sequencing gel-this separates dna fragments that differ from each other by only one base. The bands should all be the same intensity because all the different sized fragments will end with one ddntp. Automated dna sequencing (how companies sequence): Almost the same- the only difference is that dna fragments are separated by their length and running them on an acrylamide gel. They are then visualized as fluorescent peaks as the bands run off the bottom of the gel (each ddntp is a different color). This is done with a laser and fluorescent detector.