TECHNIQUES

Nota:

• increasing or decreasing the expression of a particular gene

1. Viral vector transgenesis

   Nota:

   • modifed by molecular cloning- and replicate by hijacking the protein synthesis machinery of their hosts Note dangerous- can't replicate and cause disease
   1. more efficient and versatile tools for gene delivery
   2. in-vivo/ in-vitro
   3. applicable to human gene therapy
   4. Tansgenesis

      Nota:

      • contains the coding sequence of the protein to be expressed
   5. Uses
      1. tagging proteins with fluoro
         1. conc of ions/ second messengers etc
      2. change cellular activity

         Nota:

         • by gain or loss of function
         1. function of cells
      3. Both
   6. selective tissues, organs, or cell types

      Nota:

      • e.g. using cell-specific promoters
   7. Considerations
      1. Study in a novel context needs confirming of expression- using e,g, immuno
      2. promoter activity

         Nota:

         • may vary, and some promoters are active in a wide range of cells while others only work in some cell types
      3. expression levels

         Nota:

         • some promoters take longer to build sufficient transgene expression levels, while are silenced over time
      4. immune responses- degrade transgene expression
2. Transgenic

   Nota:

   • often used to mean all types of genetic manipulations
   1. How?

      Nota:

      • random insertion of an extra piece of DNA into mouse genome
      1. 1. cDNA and promoter

         Nota:

         • you have to the cDNA of the marker/gene to a promoter of interest to relevant promoter (that drives the expression of the gene/marker)
      2. 2. inject into fertilized egg

         Nota:

         • The cDNA and promoter is integrated randomly into the genome as the genome replicates in cell division
      3. 3. implanted into mouse uterus

         Nota:

         • and transgenic pups are born if integration was successful, and are bred
   2. Considerations
      1. (+) Faster and cheaper than ES
      2. Is the phenotype a product of the gene expression?

         Nota:

         • It's important to demonstrate that the phenotype (behaviour) is a product of the expression of the targeted gene, rather than because another gene was altered/deleted as a result of the inserting
         1. Control

            Nota:

            • by demonstrating that two or more lines of transgenic mice have identical phenotypes- which will show that it's not that result of the location of the insertion of the gene, but the transgene expression
      3. Promoters

         Nota:

         • the expression of genes is dependent on the promoter, and while the authors might anticipate that they will be enough to drive the expression in target cell type, it doesn't always happen 
         1. Exogenous expression

            Nota:

            • expression of transgenes in cell types which aren't expected and haven't been checked- can affect the phenotype and 'confound' the interpretations 
         2. Control

            Nota:

            • check expression pattern in multiple cell types and tissues- even in those that aren't expected to express
      4. Position dependent expression
      5. Need to generate breed and characterise many lines
3. ES cell

   Nota:

   • targeted manipulations of specific, chosen areas of genomic DNA>> often used to delete a gene (knock-out mice) or insert mutations into a gene (knock-in mice)
   1. How?

      Nota:

      • ES cells can develop into any type of cell >> often use a hair colour gene so that you can identify which offsprings were derived from the ES cells
      1. 1. insert a sequence

         Nota:

         • into a location where is disrupts the correct expression of interest gene
      2. 2. inserted into ES cells
      3. 3. injected into blastocyst and mouse uterus

         Nota:

         • the developing embryo will have some cells derived from the original blastocyst and some from the ES cells
   2. knockout/ knockin
   3. Considerations
      1. Need to demonstrate that the gene function is altered

         Nota:

         • A strategy for testing the function of this gene must be described
      2. (-) deletion/ alteration of gene could result in death

         Nota:

         • it's important to check that the mice with the altered gene are born at a normal Mendelian ratio  which indicates that non of the mice die in utero
      3. (-) deletion/ alteration may result in animals having no phenotype

         Nota:

         • This could be due to other genes compensating for the loss of function. Important to check whether other genes are overexpressed as a result of the altered gene.  Absence of an altered phenotype doesn't mean that gene doesn't have an important function- it could be very important that there are alternatives to it
      4. (-) Genes expressed in different tissues might have a diff function

         Nota:

         • the effects in one area in the body might influence the function of the same gene at a different area
      5. (+) Can replace a murine with a human gene
4. Cre/LoxP
   1. How?

      Nota:

      • Often used to delete a gene from a specific cell type without altering expression elsewhere
      1. relies on phage ezyme Cre-recobinaze

         Nota:

         • to recognize specific DNA squences, called LoxP sites, and delete whatever is between two LoxP sites (the genomic sequence of a gene is normally too big to delete so the location of the loxP sites need to be placed strategically in order for the gene to be functionally deleted)
      2. 1. the loxP flanked animal is generated using ES cell technique
         1. 3. The two modified mice need to be crossed
      3. 2. the Cre-recombinase expressing gene is inserted using transgenic tech
   2. Considerations
      1. The two genetically modified mice need to be normal

         Nota:

         • i.e. unaffected by their genetic alteration The loxP sites/ Cre should be placed in genomic areas where they dont interfere with the normal function of the gene
         1. have the authors checked that by themselves they are similar to WT?
      2. Successful deletion?

         Nota:

         • demonstrate appropriate deletion/ dysfunction of the gene of interest
         1. PCR and immunoblot analysis
      3. 'reporter' animal

         Nota:

         • in order to evaluate where Cre is expressed throught development, animals need to be crossed with a reporter animal which expresses eGFP when a floxed transcriptional blocker is deleted by the Cre. As this process is irreversible, expression in an adult animal eGFP indicates all cells that, during their lifespan, have at some point expressed Cre
5. In pain

   Adjunto:

   • PAIN TECHNIQUES
6. USES
   1. Overexpression

      Nota:

      • overexpress a biologically active protein under the control of a unbiquitous e.g. cytokines, growth factors, neuropeptides
   2. inhibit expression

      Nota:

      • of receptors, transcriptional factors, antisense or mRNA
   3. Mark a gene (with colour)

      Nota:

      • with LacZ= blue; EGFP= green >> characterise expression pattern >> changes in expression in response to biological manipulation e.g. nerve injury, synaptic plasticity, cognitive status >> define regulatory element that control gene expression
7. synaptic

1. stimulation protocols for LTP induction

   Adjunto:

   • Techniques in synaptic plasticity

1. EEG
   1. Definition

      Nota:

      • Is a graphical representation of cortical electrical activity of the brain usually recorded from the scalp. EEG recording is state dependent and reflects ongoing neuronal activity. It changes as a function of age, arousal level, sleep stage and cerebral dysfunction.
   2. How does it work?

      Nota:

      • Small-voltage fluctuations, few tens of microvolt in amplitude, are measured between selected pairs of electrodes at the scalp. The current that generates these variations in the voltage (electrical fields) are spikes (population of action potential), EPSCs and IPSCs.

      Adjunto:

      • Techniques
      1. 4 frequency bands

         Adjunto:

         • Techniques
         1. Alpha- 8-13HZ
         2. Beta- 13-30 Hz
         3. Theta- 4-7Hz
         4. Gamma 1-3Hz
      2. Electrode placement

         Nota:

         • Note: There is no central lobe in the cerebral cortex. "C" is just used for identification purposes only. • Even numbers (2, 4, 6, 8) refer to the right hemisphere and odd numbers (1, 3, 5, 7) refer to the left hemisphere. • Z - refers to an electrode placed on the mid line. The smaller the number, the closer the position to the mid line
      3. Amplitude and Frequency

         Nota:

         • 1.Wavelength is systematically related to frequency under most circumstances because waves get shorter as frequency increases and longer as frequency decrease. 2.Faster frequencies and lower amplitudes indicates that the subject is more aroused and alert (a), whereas lower frequency, high-amplitude waves are associated with the deepest levels of sleep (b).
         1. High freq, low amp= awake
         2. Low freq, high amp= asleep
   3. Abnormalities

      Nota:

      • -Spikes –Sharp waves –Spike and slow wave complexes –May be focal, lateralized, or generalized
   4. +

      Nota:

      • 1. Non-invasive 2. Painless 3. Electrodes taped on the scalp (some three dozens) 4. Allows long-term recording (not necessarily in the clinic) 5. Relatively cheap
      1. non-invasive
      2. Allows long-term recording
   5. -

      Nota:

      • 1. Time consuming 2. Very crude (the averaging of activity from populations of neurons cannot precisely identify the brain structure generating the activity (it only establishes the brain region) EEG
      1. Cannot determine exact location of brain region
   6. Apllication
      1. Diagnosis

         Nota:

         • (epilepsy, states of altered consciousness in head trauma & coma, anoxia, intoxications, cerebral infections and sleep disorders)
      2. Monitoring

         Nota:

         • (progression and remission of brain dysfunction)
      3. Research

         Nota:

         • (use of computer driven powerful analysis of EEG patterns suggests a potential application in schizophrenia studies)
2. PET
3. fMRI
4. MEG

Nota:

• spike-triggered averaging

Adjunto:

• Techniques in synaptic plasticity

1. Extracellular recording

   Adjunto:

   • Techniques in synaptic plasticity
   1. synaptic
2. intracellular microelectrode recording
3. Voltage-clamp
4. Patch-clamp
5. synaptic

1. In pain

   Adjunto:

   • PAIN TECHNIQUES