Boiling Miniprep for Plasmid DNA Extraction

This is a rapid method for preparing partially purified plasmid DNA for restriction digestion before large-scale growth. It involves alkaline lysis to release the plasmid DNA from the cell, leaving behind bacterial chromosomal DNA and cell wall debris, and precipitation of the resulting plasmid DNA. 1. Select transformants (bacteria colonies seen on agar plate after overnight incubation) with sterile loop and place in 3-mL LB broth and the appropriate selective agent such as antibiotics (see...

Production of Retrovirus by Transient Transfections

Transient transfection methods for retroviral production are based on the high-transfection efficiency of packaging or producer systems derived from the 293 cell line, an adenovirus-transformed human embryonic kidney cell line. Transient transfection of NIH3T3-based packaging cells produces relatively low titer viral stocks (103-104 cfu mL). In addition, establishing and identifying the desired producer cells can take up to a month. In contrast, transient transfection of 293 cell-based systems...

Artifacts and Controls

When using in situ hybridization to study the expression patterns of new genes, it is crucial to include proper controls. A commonly chosen control probe is the sense strand transcript. In most cases, the sense strand is complimentary to a nonsense sequence that should not exist as a transcript in target cells. Hence, it is expected that hybridization of sense probe will not result in any specific signal. Another type of control experiment involves competing the labeled probe with nonlabeled...

Alternative Approaches

In some disorders, in both man and animals, there is increasing evidence that the metabolic defect consequent upon the mutation causes cell death through apoptosis. This would account for cone loss in patients with RP because of rhodopsin mutations (17). Apoptosis, or programmed cell death, is a genetically encoded potential of all cells, and is an essential part of embryonic development, cell turnover and of removal of cells infected by virus or harbouring mutations (18). Morphologically, it...

Preparation of RNA Probes

Single-stranded sense and antisense RNA probes are synthesized by in vitro transcription reactions using bacteriophage RNA polymerases (T7, T3, or SP6). To obtain appropriate DNA templates suitable for transcription reactions, DNA sequences encoding the probes should be subcloned into restriction sites of a plasmid flanked by two distinct bacteriophage promoters (e.g., Bluescript, Strategene). The size of the probe can range from 200 basepairs up to a few kilobases (see Notes 1 and 2). Linear...

Extraction of Genomic DNA

Different techniques for genomic DNA extraction are used, but they all involve the lysis of cells (either from tissues removed or from cell culture), deproteination, and recovery purification of DNA. When using mammalian tissue, including the retina layer gently peeled off the choroid layer, the following steps are performed 1. Remove tissue rapidly, mince and freeze tissue in liquid nitrogen. 2. Grind to a fine powder frozen tissue suspended in liquid nitrogen in prechilled mortar and pestle....

Specific Activity Determination

To determine the specific radioactivity of transcripts and oligonucle-otides, small samples (1-5 L) are spotted on glass fiber filters, dried, and analyzed in a liquid scintillation counter using scintillation fluid. Counting efficiency is presumed to be 90 . 2. For end-labeled oligonucleotides, the specific activity is determined by dividing the dpm by the concentration of oligonucleotide determined by UV absorbance. This may be converted to Ci per mole by dividing by 2.22 x 106 dpm Ci. 3. For...

Storage of Transgenic Mouse Embryos see Note

It may be desirable to store transgenic mouse embryos after the line has been characterized. To cryopreserve transgenic embryos, a transgenic male mouse should be mated to superovulated female mice of the same inbred strain. To facilitate future steps, the male mouse should be homozygous for the transgene. 1. Three days after pairing (with day of pairing considered day 0), the female mice are sacrificed and embryos are flushed from their oviducts with saline in a 5-cc syringe equipped with a...

Surgical Procedures in Mice

Avertin Stock solution dissolve 10 g 2,2,2-tribromoetha-nol (Avertin, Aldrich Chemical, Milwaukee, WI) in 10 mL 2-methyl, 2-butanol. Working solution To 1.25 mL of avertin stock solution, add PBS to a final volume of 50 mL. Store both stock and working solution wrapped in foil at 4 C. 2. Povidone-iodine 5 (Betadine 5 , Escalon Ophthalmics, Skillman, NJ). 3. 1.0 tropicamide (e.g., Mydriacyl 1 , Alcon, Fort Worth, TX) PredG (Allergan Pharmaceuticals, Irvine, CA). 4. Injection...

Electroporation of ES Cells

Once the cell lines and targeting construct are ready, electroporation and the subsequent selection procedure takes about 14 d to complete. The targeting vector is transfected into ES cells by electroporation. Generally, 25-50 g of linearized targeting plas- mid is used to transfect 2 - 4 x 107 cells. Start growing up the EF-neo cells for postpicking amplification at the time of electroporation. 1. Preparation of ES cells Thaw (see Subheading 3.3.3.) 1 cryotube of frozen ES cells (e.g., J1) 6 d...

Gyrate Atrophy of the Choroid and Retina

Gyrate atrophy (GA) is a rare autosomal recessive disorder characterized by elevated levels of serum ornithine (105) caused by reduced activity of ornithine aminotransferase (OAT), a mitochondrial matrix enzyme (106). It is supposed that high levels of circulating ornithine levels damage tissues, including the eye. Patients with GA usually have myopia and night blindness during the first decade of life, although phenotypic variation exists. Progressive visual field constriction with central...

Best Disease

Best disease, otherwise known as vitelliform dystrophy, is an autosomal dominant condition characterized by early-onset macular degeneration in the form of yellow deposits that are usually bilateral and symmetrical (see Fig. 4). Phenotypic variation is well recognized and about half of those with the abnormal gene have good vision throughout life and normal or near-normal fundi (101). Visual acuity tends to remain quite good and in some individuals may become fairly static. Choroidal...

Dehydration of Samples

Melt sufficient amount of paraffin wax in a 60 C oven. 2. Replace fixatives with 50 ethanol, immediately change the 50 ethanol to fresh 50 ethanol and incubate at room temperature for 20 min. Repeat the 20-min incubation with fresh ethanol two more times. 3. Perform the same dehydration procedure three times each using 70 , 95 , and 100 ethanol, allowing 20 min for each change. (Samples can be stored in 70 ethanol for a few days. Complete dehydration is critical for paraffin embedding.) 4....

Design of Retroviral Vectors

Retroviral vectors have been modified to accommodate nonviral genes, and usually some or all of the essential viral genes have been deleted. Therefore, the resulting retroviral vectors are often rendered replication incompetent. However, to capitalize on retroviral transcription, integration, and packaging functions, retroviral vectors usually retain the ty packaging sequence, the reverse transcription and integration signals, and the viral promoter, enhancer, and polyadenylation sequences...

Phenotype and Genotype Correlation

The outer retinal dystrophies comprise a large number of disorders characterized by progressive retinal degeneration. In the past, careful description of the clinical phenotype supplemented by functional evaluation by electrophysiology and psychophysics led to clinical categorization of retinal dystrophies into retinitis pigmentosa, macular, and rod cone dystrophies. Further subdivision was achieved on the basis of inheritance as autosomal dominant, autosomal recessive, or X-linked. However,...

Preparation of Recombinant Virus for Microinjection

HBS HEPES-buffered saline 20 mM HEPES, 150 mM NaCl, pH 7.8 filter with 0.22 mM filter. 4. Phosphate-buffered saline (PBS)-20 S 20 sucrose in PBS. PBS is prepared by dilution of a 10X stock. Composition per liter of 10X stock 80 g NaCl, 2 g KCl, 21.4 g Na2HPO47H2O, 3.5 g KH2PO4, pH 7.4. 5. Disinfectant 10 bleach (i.e., 0.525 g sodium hypochlorite 100 mL).

Derivation of Homozygous Knockout Animals

If the blastocyst injection is successful, then about 18 d after injection day chimeric progeny will be born to the foster mother. Because the blastocysts used are generally C57bl 6 embryos, wildtype progeny will have black coat color, whereas chimeric progeny will have a variable contribution of agouti coat color from the 129Sv ES cell line injected into the blastocyst. Chimeric mice with near-total or total agouti coloration are chosen for further breeding. Count up to 6 mo before homozygous...

Notes

It is important that the shuttle plasmid and the virion DNA match (see Subheading 1.). If extension transgenes are to be used then virion DNA lacking E3 sequences should be utilized. Make sure that the packaging limits of the recombined DNA do not exceed 37 kb. Purity of both the shuttle plasmid and virion DNA should be high enough (either anion column or cesium chloride purity) to ensure high efficiency of transfection. Numerous techniques for transfec-tion are now available (many commercially...

Solutions for Analysis of Proteins

4X gel buffer 1.5 M Tris base (pH 8.8), 0.4 (w v) SDS. 2. 2X stacking gel buffer 250 mM Tris base (pH 6.8), 0.2 (w v) SDS. 3. Electrode buffer 25 mM Tris base (pH 8.3), 0.1 (w v) SDS, 192 mM glycine. 4. Sample loading buffer 125 mM Tris base (pH 6.8), 4 (w v) SDS, 10 glycerol, 0.02 (w v) bromophenol blue, 4 (v v) P-mercapto-ethanol. 5. Fixing solution 50 (v v) methanol, 10 (v v) acetic acid. 6. Coomassie staining solution 50 (v v) methanol, 10 (v v) acetic acid, 0.05 (v v) Coomassie brilliant...

Picking Harvesting and Freezing Colonies

Picking of putative targeted ES cell colonies may begin on d 6 or 7 of selection. Colonies are picked from plates using an inverted microscope set up in the laminar flow hood. Clones suitable for picking are big and elevated. Colonies with a clean edge usually are the best colonies. Avoid colonies with pigmented cells (these are differentiated colonies) and pinhead colonies (these are background, non-neo resistant colonies). 3.6.1. Preparation Prior to Picking 1. Set up the inverted microscope...

Ribozyme Biology

RNA enzymes, or ribozymes, can be defined as RNA molecules that promote a variety of reactions involving RNA and DNA molecules. These include site-specific cleavage, ligation, polymerization, and phosphoryl exchange (1). The use of ribozymes for medical therapy was recognized soon after RNA catalysis was discovered in the early 1980s (2). Three broad classes, naturally occurring ribozymes have been recognized (1) RNase P, required for tRNA processing (2) self-splicing introns, including group I...

Titration of rAAVgfp

Titration of rAAV by detecting transgene expression is the most stringent method to determine rAAV titers because the virion must infect, unpackage, and express the transgene to high enough levels to allow detection. This can result in underestimates of the actual number of infectious particles. This technique is very useful with rAAV-gfp because in contrast to the figal gene, no staining is required, but just the visualization of the cells under fluorescent microscopy. However, the lack of...

Combinations of In Situ Hybridization and Immunocytochemistry

The simultaneous detection of RNA and protein expression can be accomplished by combining in situ hybridization techniques with immunocytochemistry methods. 1. Follow the in situ hybridization protocol through to the antibody blocking step (see Subheading 3.6.2., step 4 or Subheading 3.7.2., step 5). The in situ antibody blocking solution (10 sheep serum TBST) can be supplemented with 2 normal serum from the species of secondary antibody that will be used to detect the primary antibody. For the...

Amplification Purification Large Scale Adenoviral Preparation

Split rapidly growing 293 cells 1 4 or 1 5 and place finally in 24-30-vented triple flasks at approx 70 confluency (see Note 8). 2. Transduce with either purified virus or CVL at an MOI of 0.05-0.1. 3. Transduce the 293 cells in 20 mL of IMEM containing 2 FBS. Place in incubator and wait 90 min. Add 20 mL of IMEM containing 20 FBS. 4. Carefully watch cells and look for beginning of CPE, which typically occurs within 24-48 h (see Note 9). Cells are typically ready to harvest after approx 48-72...

Determination of fiGalactosidase Activity and Quantification of Transcription Levels

The luciferase activity measured following transient transfections must be normalized for transfection efficiency and for general effects on transcription using an internal control vector (see Note 10). Therefore, all cells are routinely cotransfected with a control plas-mid containing the bacterial lacZ gene driven by the SV40 early promoter, the pSV- -galactosidase control vector (Promega). The method of Sambrook et al. is used (15) with minor modifications. 1. Pipet 40 L of 4 mg mL ONPG...

Purification of Ribozymes and Target RNA

Ribozymes and target RNA molecules produced by in vitro transcription may require purification by gel electrophoresis to assure homogeneity. Ribozymes or larger targets (> 30 nucleotides) can be purified on 8 acrylamide, 8 M urea sequencing gels run in TBE buffer (see Subheading 2.4. 4,32 ). 2. Labeled molecules are visualized by autoradiography, excised with a sterile scalpel, and eluted from the gel in elution buffer (see Subheading 2.4., step 5) for 1-4 h. 3. If gel purification is...

Denaturing Gradient Gel Electrophoresis DGGE see Note

Design PCR Primers as in Subheading 3.2., step 1a-e with the following exceptions a. Include a large GC clamp (24) on the 5' end of the primer with the most G's and C's. One example is GCC GCC CGC CCG CCC GCC GCG CCG CCC GCC GCC CGC (18). This primer will then be 18-24 bp plus the 36 bp GC clamp the other primer remains at 18-24 bp. b. Select primers to amplify a fragment of approx 300 bp (the GC clamp adds 36-40 bp). Much more than this decreases the sensitivity of the DGGE method for...

Blastocyst Injection

Because of the high investment required in sophisticated micromanipulation apparatus and the expertise to use it, it is highly recommended that the investigator contract this out to a suitable institutional core facility or commercial provider. Many academic centers provide this service. Commercial providers include Genome Systems Inc (St. Louis, MO). 1. Preparation of targeted ES cell line for blastocyst injection 2. Set up EF-neo cell cultures in 6-cm dishes and grow to confluency in 2-3 d...

Labeling of RNA Oligonucleotides

Protecting groups are removed from the 2' positions of RNA oligonucleotides according to the manufacturer's instructions. 2. RNA oligonucleotides (typically, 14mers) are assembled with the labeling reagents (see Subheading 2.3., step 6). 3. The mixture is incubated at 37 C for 30 min and then is diluted to 100 L with sterile water and extracted with phenol chloroform isoamyl alcohol (50 50 1) to inactivate the enzyme. 4. Unincorporated nucleotides are removed by passing the aqueous phase over a...

Hybridization of Probes and Washing

The hybridization of nucleic acid molecules in fixed tissues is a complex process, and at present is not fully understood. Therefore, hybridization conditions are largely tested empirically. The melting temperature (Tm) of a given nucleic acid hybrid depends on the extent of homology between the probe and the target, the length of the probe, the base composition of the probe, and the type of hybrid formed (RNA-RNA, RNA-DNA, or DNA-DNA). In addition, the Tm of a nucleic acid hybrid in tissue is...

Preparation of Monolayer Cells

Coat glass slides with 10 g mL poly-D-lysine at room temperature for 30 min. Rinse the slides once with PBS, and set horizontally in a moist chamber. 2. Pipet suspensions of cells (2 x 107- 1 x 108 cells mL) onto a slide as 10 to 100 L drops. Place slides in a 37 C, 5 CO2 tissue-culture incubator for 1 h to allow cell attachment. Alternatively, one can directly culture cells on the slides for prolonged periods. 3. Fix cells by submerging slides in fresh 4 PFA at room temperature for 10 min. 4....

CotransfectionAd Infection of293 Cells

Generation of rAAV-gfp virus involves cotransfection of 293 cells with the recombinant plasmid SSVgfp (Subheading 3.1.2.) and the helper plasmid pAAV Ad, followed by adenovirus infection (see Note 2). A schematic representation of the protocol is provided in Fig. 2. The 293-cell line, which constitutively expresses E1 proteins, is used to make AAV recombinant virus because they allow a good efficiency of transfection and the use of E1-deficient adenovirus (replication defective) as helper virus...

Preparation of EF Cells for ESCell Culturing

The methods described below are based on those of Robertson (15). While ES cells have been successfully grown on plastic (16) in the presence of leukemia-inhibitory factor (LIF), they grow better on a feeder layer of embryonic fibroblast (EF) cells. LIF is a differentiation-inhibiting cytokine necessary to prevent differentiation of ES cells (17). Primary EF cells produce some of this factor, but it has to be supplemented with recombinant LIF (ES-gro, Gibco, Gaithersburg, MD). EF feeder cells...

References

H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156. 2. Doetschman, T., Gregg, R. G., Maeda, N., Hooper, M. L., Melton, D. W., Thompson, S., and Smithies, O. (1987) Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576-578. 3. Bradley, A., Evans, M., Kaufman, M. H., and Robertson, E. J. (1984) Formation of germ-line chimeras from embryo-derived teratocarci-noma cell lines. Nature 309,...

The Life Cycle of a Retrovirus

Naturally occurring retroviruses are replication competent. Upon infection of a target cell, the viral genome is replicated and packaged into viral particles that can infect new host cells. Retroviral infection occurs through an interaction between viral particles and specific receptor proteins on the surface of the target cell. Upon entry into the target cell, the retroviral RNA genome is reverse transcribed into DNA by a viral encoded reverse transcriptase (RT) enzyme (Fig. 1A). The duplex...

Solutions for Analysis of DNA

Tris acetate (TAE) buffer 40 mM Tris-HCl (pH 8.0), 1 mM EDTA. 2. Tris borate (TBE) buffer 89 mM Tris-HCl (pH 8.3), 89 mM boric acid, 2 mM EDTA. 3. DNA loading buffer 25 (w v) Ficoll 400 or 50 (w v) sucrose, 100 mM EDTA, 0.1 (w v) bromophenol blue. 4. Denaturation buffer 1.5 M NaCl, 500 mM NaOH. 5. Neutralization buffer 1.5 M NaCl, 500 mM Tris-HCl (pH 7.0). 6. Transfer buffer 20X SSC 3 M NaCl, 300 mM trisodium citrate or 0.4 M NaOH.

ES Cell Culture

First, a frozen aliquot of a suitable ES cell line (for example, J1 see ref. 4) should be obtained. This should be stored in liquid nitrogen until required. For the cell culture techniques described here, an adequately equipped tissue culture laboratory is necessary. Essential equipment includes 37 C tissue culture incubators set to 86 humidity and 5 CO2, a laminar flow biological safety hood, a table-top clinical centrifuge, an inverted microscope, and a liquid nitrogen repository. The...

Precipitation of rAAV and Ad

Before banding on a CsCl gradient, the virus needs to be concentrated and most of the cellular proteins removed. For this purpose, two successive precipitations with (NH4)2SO4 are performed. The first one will precipitate the undesired proteins and the second will precipitate the rAAV virions and adenovirus. 1. Pool the supernatant and add 1 3 vol of cold saturated (NH4)2SO4. Mix well and put on ice for 10 min. Typically, this would be 20 mL of supernatant + 7 mL of (NH4)2SO4. The saturated...

Delivery and Uptake of ODNs In Vitro and In Vivo

As the effect of ODNs will largely depend on their ability to enter into the cells, it is essential to establish if the target cells are able to take up and retain ODNs at a concentration suitable to induce a biological effect. In this section, an uptake study will be described by quantification of the uptake of an FITC-labeled ODN by retinal pigment epithelial (RPE) cells (see Note 3). Average of three measurements. Average of three measurements. 1. Subculture frozen RPE cells (American Tissue...

Production of Retroviruses

The production of replication-incompetent infectious retroviral particles relies on the supply of essential viral proteins from an exogenous source. Towards this end, various packaging cell lines stably transduced to express of essential viral proteins are established. The most commonly used packaging cell lines are derived from human 293 cells, primate COS cells, mouse NIH 3T3 cells, and quail QT6 cells. During viral production, recombination events may lead to wild-type (helper) viruses....

Posthybridization Washes and Antibody Binding Day 2

Carefully lift cover slips from slides or remove slides from slide mailers, and place individual slides directly into prewarmed washing solution I in a slide tray or a Coplin jar. 2. Wash three times for 15 min each in washing solution I at 70 C. 3. Wash three times for 15 min each in washing solution II at 65 C. 4. Wash three times for 10 min each in TBST at room temperature. 5. Block in 5 sheep serum TBST, for 1 h at room temperature, or overnight at 4 C. 6. Dilute the antidigoxigenin...

Analysis of DNA

Agarose Gel Electrophoresis Agarose gel electrophoresis is useful for separating DNA fragments. Minigels are good for rapid separation of small amounts of DNA for quick analysis of restriction digestion. The larger scale gels are used for longer electrophoresis for better resolution of DNA fragments and are well suited for Southern blotting. 1. Add appropriate amount of electrophoresis grade agarose to electrophoresis buffer (see Note 3). The electrophoresis buffer generally used is...

Autosomal Dominant RP adRP

The reported relative incidence of adRP ranges from 10 to 25 . The adRP group is reported on average to be milder and have a later Appendix of All Known Loci of Retinal Disorders to Date (May 1999) (30) 1p31 Recessive Leber congenital amaurosis recessive RP 1p21-p13 Recessive Stargardt disease, juvenile and late onset recessive MD recessive RP recessive fundus flavimaculatus recessive combined RP and cone-rod dystrophy 1q13-q23 Dominant RP 1q25-q31 Dominant MD, age-related 1q31-q32 Recessive...

Host Ranges of Retroviral Vectors

The host range of a retrovirus particle is determined by the viral glycoprotein env and host cell surface receptors. Murine and avian retroviral vectors have several types of env proteins that interact with different host receptors. Murine ecotropic retroviruses bind to the ecotropic receptor found on rat and mouse cells, and do not infect human cells. The amphotropic env proteins, however, endorse a murine retrovirus with a broader host range including rodents, human, chicken, dog, and cat....

Immunofluorescence Processing of Cryo and Vibratome Sections

Preparation of Cryosections 1. Fix retinal samples for 6 h or longer in 4 paraformaldehyde in 0.13 M phosphate buffer, pH 7.3. 2. For glutaraldehyde-fixed tissue, use sodium borohydride treatment. 3. Soak tissue overnight in 30 sucrose in phosphate buffer at 4 C. 4. Freeze tissue in OCT (Polysciences) on a cryostat chuck. 5. Section at 8-14- m thickness. 6. Mount sections on subbed glass slides. 7. Air-dry overnight at RT. Slides can be stored at -80 C. 3.1.2. Sodium Borohydride (NaBH4)...

Congenital Stationary Night Blindness CSNB

CSNB is a heterogeneous group of disorders characterized by poor night vision that is life-long and described as nonprogressive. Dark adaptation may be absent or markedly prolonged. X-linked inheritance is the most common pattern in CSNB. Autosomal recessive and dominant forms are also seen. Oguchi disease and fundus albipunctatus are distinct forms of CSNB. CSNB is usually classified into two distinct groups according to the electrophysiological findings. In type I, there is no rod a-wave...

Determination of Retroviral Titers see Note

Titers of retroviral stocks can be determined by using viral encoded selectable drug-resistant markers or histochemical markers (12). The murine BAG virus expresses both the neomycin resistance gene and the lacZ gene, and thus can be titered with either method (11). Titers of avian viruses can also be determined by immunocytochemistry using an antibody that recognizes the viral gag protein (22). 1. Split NIH3T3 target cells 1 10 or 1 20 into the wells of a 12-well dish the day before infection...

Pretreatment and Hybridization

Rehydrate samples stored in 100 methanol by sequential 5-min washes in 75 , 50 , 25 methanol PBT, followed by two washes with PBT. 2. Bleach samples with 2 H2O2 in PBT for 1 h at room temperature, followed by three washes with PBT. 3. Treat samples with 10 g mL proteinase K in PBT for 15 min at room temperature while rocking. a. The concentration and duration of proteinase K treatment are critical, and must be empirically determined. For younger embryos or ectodermal detection, less proteinase...

Ribozyme Cloning and Labeling

DNA oligonucleotides encoding the ribozyme sequences with appropriate flanking restriction sites can be ordered from a variety of commercial sources. 2. Large fragment of DNA pol I (Klenow) and DNA ligase are purchased from New England Biolabs (NEB). 3. Unlabeled nucleoside triphosphates should be obtained from a high-quality vendor and purchased as a buffered stock solution (typically, 200 mM). 4. Transcription vectors include pT7T3-19 (Gibco-BRL) and pBluescriptII KS+ (Stratagene). 5. RNA...

Genotype Phenotype Correlations

To dilate mouse eye 1 tropicamide (e.g., Mydriacyl 1 , Alcon, Humacao, Puerto Rico). 2. Slit Lamp (Carl Zeiss, Germany sold through Krebs, Englewood, NJ). 1. Dilating drops (see Subheading 2.5.1.) Ophthalmoscope Heine Omega 180 binocular indirect, Krebs, Englewood, NJ) and 78 or 90 diopter lens (Krebs). 2. Fundus photography Kowa Camera (Krebs, Englewood, NJ) and 78 or 90 diopter lens. 1. Cryosections Selection of fixative is dependent on what additional studies are desired. The fixative least...

Separation of Proteins on Sdspage

This involves heat denaturation of the proteins in the presence of SDS and a reducing agent such as p-mercaptoethanol or dithio-threitol (DTT) to reduce disulfide bonds. The SDS coats the proteins, giving them a negative charge proportional to their length. On application of an electric field, the proteins separate by charge and by the sieving effect of the gel matrix. The separation of the proteins can be enhanced using a discontinuous gel system that has stacking and separating gel layers...

Extraction of Nucleic Acid

Genomic DNA and RNA are used for preparation of genomic or complementary DNA (cDNA) libraries, respectively. Genomic DNA is also frequently used for mapping of genes, and total or messenger RNA (mRNA) is normally used for gene expression studies. Genomic DNA fragments or cDNA from transcription of total RNA are often cloned into plasmid vectors for further analysis or manipulation (Subheading 3.4.). Currently, kits are available from a number of companies for nucleic acid extraction, but the...

Assaying Excision and Replication of Viral DNA

Many different types of promoters and gene cassettes are being cloned in AAV vectors and tested in eukaryotic cells. Although still limited, some sequences have been found to be incompatible with AAV vectors. The ability of the recombinant genome to be rescued and replicated during the complementation process is easily tested by analyzing low-molecular-weight DNA isolated from transfected, adenovirus coinfected cells. This protocol is a modification of the Hirt procedure (15). 1. Collect...

Isolation of DNA from Blood Leukocytes

Lysis buffer 111.25 gm sucrose, 5 mL 1 M MgCl, 10 mL Triton X-100, 10 mL 1 M Tris-HCl pH 7.8, make to 500 mL with water. Store at 4 C. 2. Saline ethylenediamine tetraacetic acid (EDTA) 2.19 gm NaCl, 4.56 gm EDTA, make to 500 mL with water and autoclave. 3. Protease K solution Use Sigma P-0390 protease K and make to 20 mg mL in water. 4. 20 sodium dodecyl sulfate (SDS) Dissolve 400 gm sodium lauryl sulfate in water and bring to 2 L. Mix with a mag stirrer overnight. Filter sterilize. 5. Phenol...

Total Retinal RNA Extraction

Total RNA from the retina is conveniently recovered by extraction with Trizol according to the manufacturer's recommendations. Retinas can be snap frozen in liquid nitrogen prior to RNA extraction and stored at -70 C. A single mouse or rat retina is resuspended in 1 mL Trizol reagent and homogenized by repeated passage though an 18-gage syringe needle. 2. Following a 5- to 15-min incubation at room temperature, the suspension is extracted with 0.2 mL of chloroform. 3. After centrifugation to...

In Vivo Assessment by Ophthalmoscopy see Note

Ophthalmoscopy can be used to verify that the retina has been detached immediately after subretinal injection and that it has reattached during the recovery period. Ophthalmoscopy can also be used to assess the health of the retina and to identify fluorescently tagged transgenes through illumination with the exciting wavelength of light (1,6). 1. Dilate the pupils as described in Subheading 3.2.1. or 3.3.1. The rodent fundus can be observed under illumination from the light of the indirect...

Identification of Transgenic Mice

Obtain a biopsy from the tips of mouse tails using a sterile razor blade. 2. Digest the tails overnight at 55 C in tail buffer with proteinase K at 0.5 g L. 3. Isolate genomic DNA using a series of phenol and chloroform extractions. 4. Precipitate and rehydrate DNA in TE pH 8.0. 5. Digest genomic DNA (20 g) with selected restriction enzymes and subject to electrophoresis. 6. Transfer DNA to a nylon or nitrocellulose membrane and perform Southern blot analysis. 7. Use a 32 P -radiolabeled probe,...

And Longer Target RNAs by In Vitro Transcription

Plasmid DNAs containing target sequences or ribozyme genes are linearized with a restriction enzyme that cuts downstream of the sequence to be transcribed. 2. Endonucleases and contaminating ribonucleases are inactivated by extraction with phenol-chloroform-isoamyl alcohol (50 50 1) followed by ethanol precipitation. 3. Transcripts are generated with T7 (or other) RNA polymerase and labeled by incorporation of a- 32P -UTP using the protocol of Grodberg and Dunn (30). A 100- L reaction (see...

Harvesting the Cotransfected AdInfected Cells

Transfer the cotransfected Ad-infected 293 cells and media (Subheading 3.1.5.) to centrifuge tubes, and pellet at 500g, for 5 min. 2. Resuspend the pellet in PBS at 1 mL plate. 3. Sonicate cells in three 20-s bursts on ice (output control 5, duty cycle 50 ). Because AAV accumulates in the nucleus of infected cells, this step is important to disrupt the cells nucleus completely. 4. Vortex the lysate at top speed for 30 s and spin at top speed in a tabletop centrifuge for 10 min.

Introduction

Adenoviruses are viruses with genomes of double-stranded DNA approx 36 kb in size. There are more than 100 different serotypes that have been identified, 47 of them of human origin. Human serotypes have been grouped into six subgenera (A-F) and whereas sequence similarity between genera is low, other molecular biologic aspects of the viruses are almost identical. Of all the human adenoviruses, serotypes 2 and 5 (subgenus C) have been the most extensively studied (1). Recently, more interest in...

Visualization of Proteins Resolved on

Coomassie Brilliant Blue Staining This method depends on the nonspecific binding of the dye to proteins and the limit of detection is between 0.3 and 1 g protein per band. 1. Remove gel from plate and place in container. Cut corner of gel for orientation. Add five gel volumes of fixing solution. Gently rock gel for 2 h in orbital shaker. 2. Remove fixing solution and replace with fresh fixing solution with 0.05 (v v) Coomassie brilliant blue added. Leave for 2-4 h. 3. Remove solution...

Single Strand Conformation Polymorphism Electrophoresis SSCP see Note

Design PCR primers from the sequences flanking each exon according to the following criteria. a. Select primers that amplify from at least 20 bp in front of the exon to 60 bp after the exon. This is to include the entire 14 bp acceptor splice sequence upstream of the exon, and the 14 bp donor splice site downstream of the exon plus possible branch point sites at 30-50 bp downstream of the exon. b. Select front and rear primers that have approximately the same percentage of G C bases. This is to...

Positive Negative Selection

Label the 60 6-cm EF-neo plates one plate with ES and ESM, one plate with ES and G418, two plates with Elec. ES and G418 and all the others with Elec. ES with G418+Gan. 2. Resuspend the electroporated cells with ESM to a density of 2.5 x 105 cells 4 mL and add 4 mL of ESM and cells to all of the 6-cm plates marked with Elec. ES. 3. Add nonelectroporated ES cells (from the aliquot removed for counting) to the other control plates marked with ES. 4. 24 h later, start the single (G418) and double...

Leber Congenital Amaurosis LCA

Early onset severe retinal degenerations are known as LCA and are classically inherited as autosomal recessive disorders. Nonallelic heterogeneity has been proved in LCA with mutations in the RETGC (67), RPE 65 (68), and CRX genes (69). Most of the LCA patients with RETGC and RPE 65 mutations are homozygotes or compound heterozygotes. De novo mutations have been reported in RPE 65 and CRX that may indicate dominant disease (70,71). Little is known of the disease mechanisms, except for RPE 65,...

Screening of Putative Targeted ES Ceil Clones

Preparation of Genomic DNA from ES Cells (18) 1. Make up lysis buffer 100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2 sodium dodecyl sulfate (SDS), 200 mM NaCl, 100 g proteinase K mL. 2. After ES cell clones in the 24-well dish reach confluency (see Subheading 3.6.4.), remove media and replace with 0.5 mL of lysis buffer. Return the plate to a 37 C incubator overnight. 3. In the morning, add 0.5 mL of isopropanol and agitate on a rocking platform or orbital shaker until it is well mixed and the...

Multiple Turnover Kinetic Analysis see Note

Analyses to determine multiple turnover kinetic constants are typically carried out in 20 mM MgCl2, 40 mM Tris-HCl, pH 7.5, at 37 C for short intervals. The appropriate interval is determined by a time-course experiment under multiple turnover conditions (i.e., substrate excess). Initial rates should be measured when the amount of cleavage is linear with time and when no more than 10 of substrate has been converted to product. Rates are measured at several intervals (e.g., 5, 10, and 20 min) to...

Selection of Antisense ODN Sequence

Analysis of Target DNA Sequence For the selection of antisense ODNs, it is necessary to download either the genomic or cDNA sequence of the target DNA. Both the genomic and cDNA sequences preferably should contain some of the 5' untranslated region. The genomic DNA sequence is usually long 10-20,000 bp. The cDNA sequence is derived from the mRNA, thus it only contains the exons. Exons are the parts of the genomic DNA that are translated into protein. Generally, the cDNA sequence usually...

Detection of Wild Type Recombinant Virus see Note

Plate A549 cells in a 150-mm dish in A549 complete media. 2. When cells are at 80-100 confluency, place vector stock in 5 mL of serum-free DMEM at an MOI 10-20 (approx total of 5 x 108 pfu of viral vector). 3. Place on cells for 90 min then add 5 mL of A549 complete media. 4. Replace media at 24 h and every 3 d with A549 complete media. 5. By 5-6 d, cell changes should be evident, which are a result of direct viral penton toxicity. 6. Scrape cells and pellet by centrifugation at 2000g at 4 C...

RTPCR Analysis of Ribozyme Cleavage Products from Full Length Retinal RNA

To distinguish wild-type opsin mRNA from mutant transgene mRNA, we use a three-primer system for PCR amplification. The downstream primer anneals to both the mutant and wild-type cDNA and two separate upstream primers anneal to either the mutant or wild-type cDNA (34). This reaction results in PCR products of different lengths from wild-type and mutant mRNA. 2. RT-PCR reactions initially contain 1 g of total retinal RNA, 1 pmole of primer, Superscript first-strand buffer (Gibco-BRL), and 200 m...

Culture of Embryonic Stem ES Cells

These are general techniques to be followed for the culture of ES cells. 1. Refeed cells 2-3 h prior to subculturing. 2. Aspirate the medium and wash two times with 5 mL of H H. 3. Flood the plate with 1 mL 0.25 HEPES-trypsin-EDTA. 4. Incubate at 37 C for 2.5 min. 6. Incubate at 37 C for 2.5 min. 7. Agitate with 1-mL pipet 10x and check under microscope. 9. Agitate 15 times with a 5-mL pipet. 10. Transfer to 15-mL tube and add 5 mL ESM. 11. Spin 3.5 min on setting 3 using a tabletop clinical...

Preparation of Whole Mount Embryos and Tissues

Dissect embryos or tissues in cold PBS. Transfer samples to scintillation vials containing 15-30 mL of fresh 4 PFA, and fix at 4 C overnight with gentle rocking (see Note 9). 2. Remove the fixative and wash samples in 5-10 mL PBT twice for 5 min each at 4 C with gentle rocking. 3. Dehydrate samples by washing in 25 , 50 , 75 methanol PBT sequentially, 5 min each at room temperature with gentle rocking. Then wash two times with 100 methanol for 5 min each with gentle rocking. 4. Dehydrated...

Pseudotyping Retroviruses with Vsv G Protein

Retroviruses can be pseudotyped with VSV G protein to broaden the host range. VSV G is fusogenic and thus toxic to cells when stably expressed. However, transient transfection of cells with a VSV G expressing plasmid allows incorporation of the VSV G protein into viral particles before the onset of massive cell death. Stable viral producer lines can be transiently transfected with a VSV G expressing plasmid, or packaging cell lines can be cotransfected with VSV G plasmid and a retroviral vector...

Reagents and Solutions

Helper virus plasmid DNAs Ecotropic helper DNA (19,20), or ampho-tropic helper DNA (19,20). 3. VSV G protein expression plasmid, e.g., pHCMV-VSV-G (16). 4. Culture media For 293 cell and its derivatives, use Dulbecco's modified Eagle's medium (DMEM, high glucose, 2 mM L-glutamine) containing 10 fetal calf serum (FCS) for NIH 3T3 cell and its derivatives, use DMEM containing 10 calf serum (CS). 5. HEPES-buffered saline (HBS) 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose, 21 mM HEPES....

Other Useful Methods

Isolation of Genomic DNA from Tail Biopsies Genomic DNA is prepared from tail biopsies by a simple salting-out procedure 1. Place each 1-cm tail biopsy in 500 L lysis buffer (200 mM NaCl, 20 mM EDTA, 40 mM Tris, pH 8.0, 0.5 SDS, 0.5 2-p-mercapto-ethanol, 400 g mL proteinase K) and lyse overnight at 55 C. 2. Vortex each tube for 10 s. Add 250 L of saturated NaCl (about 6 M) and vortex another 10 s. Place on ice for 15 min. 3. Spin the tubes at maximum speed in a microfuge. 4. Transfer...

Avidin Biotin Methods

A second useful technique is based on the same principle, but instead of using a fluorochrome, employs a secondary antibody labeled with biotin, followed by application of avidin, then of biotin labeled with horseradish peroxidase (HRP). This technique, includ- Fig. 2. Illustration of ABC technique. The outer layers of rabbit retinas were incubated over wells containing PBS with or without a biotinylated protein tracer, followed by avidin-HRP to demonstration diffusion of the tracer. Left panel...

Neo Gene Specific Polymerase Chain Reaction PCR

The presence or absence of the neo gene can be assayed for by neo-specific PCR using primers recommended by the Jackson Laboratory Induced Mutant Resource (8). These are 1. oIMR013 (5'-CTT GGG TGG AGA GGC TAT TC-3' Tm 58.7 C) 2. oIMR014 (5'-AGG TGAGAT GAC AGG AGA TC-3' Tm 53.9 C). Use these in a reaction consisting of 5. 0.25-0.5 U Taq DNA polymerase.

Transient Transfections of Human Rb Cells Using Calcium Phosphate Precipitation

2x HEPES-buffered saline Na2HPO4, pH 7.0 (HBS P) 45 mMHEPES (tissue-culture grade), 280 mM NaCl, and 2.8 mM Na2HPO4. Adjust pH accurately to 7.0 with NaOH, filter sterilize. This buffer may be stored at 4 C for several months recheck pH after prolonged storage. 2. Phosphate-buffered saline (PBS), pH 7.4 140 mM NaCl, 2.7 mM KCl, 4.5 mM Na2HPO4, and 1.5 mM KH2PO4. Adjust pH to 7.4 with HCl, filter sterilize. 3. 2.5 M CaCl2 (tissue-culture grade), filter sterilize. 4. 10x TE buffer, pH 7.0 10 mM...

Isolation of mRNA

Polyadenylated or poly(A)+ RNA species (most eukaryotic mRNAs) represent only a small fraction of total RNA. Poly (A)+ RNA can be purified from nonpoly (A)+ RNA (rRNA and tRNA) using oligo(dT) cellulose. This method relies on the binding between the poly(A)+ residues on the 3' end of the mRNA and oligo(dT) residues coupled to the cellulose column matrix. The unbound RNA is then washed off the column and the poly (A)+ RNA is eluted by lowering the amount of salt in the column buffer. Poly (A)+...

Solutions for Immunogold Procedure

Blocking buffer for light microscopy 100 mL of PBS, 0.3 mL of 10 Triton X-100, 1 mL of horse serum, 1 gm of BSA. 2. Tris-buffered saline (TBS, 20 mM) 900 mL of dH2O, 9 gm of NaCl, 2.42 gm of Tris base (tris hydroxymethylaminomethane), adjust pH to 8.2 with concentrated HCl. 3. BSA-TBS 90 mL of TBS, 0.1 gm of BSA, 0.5 mL of 10 Triton X-100, add dH2O to 100 mL, filter through 0.2 m membrane filter immediately before use to remove microorganisms and particulates. 4. BSA-TBS-NaCl 50 mL of BSA-TBS,...

Whole Mount In Situ Hybridization Figs 1 and

The protocol described below is derived from the method used by Riddle et al. (4-7). All washes are performed in vials with 5-10 mL of solution at room temperature for 5 min with gentle rocking on an Orbitron rotator, unless otherwise noted. Fig. 2. Section in situ hybridization of chick retina with Pax6 RNA probes. Micrographs of chick eyes processed for section in situ hybridization as described in this chapter are shown. Cryosection of 15- m thickness were hybridized to antisense chick Pax6...

Electron Microscopic Immunocytochemistry

For electron microscopic (EM) localization of antigens, the retina can be labeled before or after the tissue is embedded in EM resin. 1. Process free-floating pieces of tissue, cryo- or vibratome sections as above by the ABC technique using DAB as the chromogen. 2. Postfix tissue in osmium tetroxide or gold chloride (7), embed in an epoxy resin, and cut ultrathin sections for EM. 3. Examine and photograph sections unstained or lightly contrasted with lead citrate. Advantages of this technique...

Detection and Visualization of Bound Probes

Radioactive in situ hybridization signals are detected by overlaying tissue sections with a light-sensitive photographic emulsion. The radioactive signals are captured as reduced silver grains in the emulsion layer and visualized using light microscopy. 35S-labeled probes are commonly used because the hybridization signals are easily detected and relatively defined (Table 1). Exposure times Summary of Labeling and Detection Methods Used for In Situ Hybridization a. Direct detection of...

And Measurement of Luciferase Activity

It is accepted that the level of luciferase activity measured following transient transfection correlates well with the level of the luciferase reporter gene expression. In order to prepare cell lysates suitable for luciferase activity measurements, the luciferase assay system (Promega) is used according to the manufacturer's instructions with minor modifications 1. Aspirate the medium from the plates and wash the cells twice with 4 mL of PBS at room temperature (see Note 8). 2. Add 0.1 mL of...

Detection and Visualization of Hybridized Probes

Wash three times in NTMT, each for 10 min at room temperature. 2. Incubate with freshly made X-Phos reaction mix. Cover tubes with foil, place on rocker at room temperature. Check on reaction progress after 20 min, and then monitor about once an hour, as necessary (see Note 12). 3. When reaction is judged complete, wash two times in NTMT for 10 min each, rocking in dark. 4. Wash two times in PBT (pH 5.5), for 10 min each at room temperature. 5. Postfix with 4 Paraformaldehyde and 0.1...

Sorsbys Fundus Dystrophy SFD

Sorsby and Mason described four families with autosomal dominant macular dystrophy (119). The fundus features are similar to those of exudative type of age-related macular degeneration (AMD), but patients present at a younger age (see Fig. 6A,B). Central visual loss is usually secondary to choroidal neovascularization (CNV) in the fifth decade (120) or later because of geographic atrophy. SFD was linked to chromosome 22q13ter (121) and subsequently a point mutation was identified in the tissue...

Single Turnover Kinetic Analysis

The rate of the cleavage step of a ribozyme (kobs), as distinct from the overall rate depending on association and dissociation steps, can be determined in ribozyme excess experiments. This is often a useful statistic in comparing a new ribozyme with others that have been used successfully in the past. 2. The relative amounts of ribozyme and substrate must be experimentally determined, but 20 1 is a reasonable starting condition. (Rates should not vary with ribozyme concentration if an adequate...

Constructing the Targeting Vector

Cloning and Sequencing of the Gene to be Targeted Prior to embarking on a gene-targeting project, it is to be expected that the investigator has already acquired significant information about the gene of interest. This could include information about tissue expression, developmental expression, and protein localization secreted, membrane, or cytoplasmic . Information about the possible function of the protein expressed by the gene will also assist in the ultimate analysis of phenotype is...

Reagents Solutions and Recipes

1. 10X phosphate-buffered saline 10X PBS , pH 7.3 Dissolve 80 g NaCl, 2 g KCl, 11.5 g Na2HPO47H2O, and 2 g KH2PO4 in double-distilled dd H2O to a final volume of 1 L. Titrate to pH 7.3 with 1 N NaOH. 2. 30 Sucrose PBS Dissolve 30 g of sucrose in 1X PBS to a final volume of 100 mL. Filter and store at 4 C. 3. 10 mg mL poly-D-lysine solution Dissolve 1 g of poly-D-lysine high molecular weight, Sigma in 100 mL of dd H2O. Filter, and store in aliquots at -20 C. Dilute to 10 g mL with H2O before...