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Dr. Anand Srivastava: Excellence in Biological Techniques

Techniques related to Human Embryonic Stem Cell

Human Embryonic Stem cell culture, Serum-free and feeder-free hES cell culture, in vitro differentiation of hES cells into neural cells, in vitro differentiation of hES into hematopoietic cells and red blood cells under the control of cytokines. Gene regulation studies using RT-PCR, Real-time PCR, Northern blot, Southern blot and in situ hybridization, immunohistochemistry during the differentiation, Cell cycle regulation studies during differentiation of hES cells into hematopoietic and neural cells. Use of siRNA for blocking a specific cell cycle. FACS analysis of differentiated cells and cell shorting. ES cell transfection.

In vivo studies with ES cells

Created a mouse model for studying the effect of ES cells on a damaged brain. Injection of ES cells into a mouse brain, tail vein injection, in vivo tracking of ES cell migration. Used the ES cells for repair of damaged brain. Gene and protein regulation during neural cell differentiation. Studies on transcription factors. Histochemical analysis of transplanted ES cells using fluorescent, confocal microscopy and deconvolution microscopy.

Created a mouse model for Crohn’s disease. In vivo migration of ES cells into diseased portion of intestine. Studies on inflammatory cytokines during the repair of Crohn’s disease with ES cell. Gene regulation studies during this process. Elisa assays for the cytokines. Stem cell niche interaction.

Created in utero mouse model for ES cells transplantation. Used this model to make chimeric animals. Distribution and differentiation of ES cells into developing mouse embryo. FACS and magnetic shorting of ES cells derived CD31+, CD34+, CD45+ cells from the transplanted animal tissues. Gene and protein regulation of in vivo differentiating cells.

Created an immunocompromised mouse model to study the effect of in vivo immune component on T7 phage virus. In vivo selection of tissue-specific receptor binding peptide using in vivo biopanning method. Tissue targeted gene delivery to correct the blood related genetic diseases. Gene cloning, gene sequencing, synthesis of RNA probes.

Protein and enzyme biochemistry

Protein assay, peptide structure, amino acid sequencing, Enzyme assay, Ultracentrifugation, Ion exchange chromatography, column chromatography, HPLC, and Protein and gene regulation during the development. Enzyme kinetics, Enzyme inhibition, SDS gel electrophoresis, Protein characterization.

Selection of cell receptor binding peptide and Phage display technology

  • Selection of tissue receptor binding peptides using T7 phage display system.
  • In vivo and in vitro biopanning for selection of receptor binding peptides sequences.
  • Characterization of targeted cells and tissues using histochemistry and gene expression analyses.
  • In vivo delivery of drugs and genes to targeted tissues using microinjection.

Cancer Research 

  • Studying the role of pharmaceutical agent curcumin as an anti-lung cancer drug and development of the agent as a non-toxic cancer drug.
  • Role of apoptotic genes on the lung cancer cell lines.
  • Development of tissue targeted delivery protocol of pharmaceuticals agents for cancer and genetic diseases.

Fluorescence techniques for nucleic acid sequence detection: clinical and diagnostic applications

  • Fluorescent labeling of DNA and RNA probes.
  • Fluorescence Resonance Energy Transfer (FRET) protocols for DNA and RNA sequence.
  • Detection in real time (Sequence Detection System 7700, ABI, Perkin Elmer).
  • FRET protocols for monitoring ribozyme reactions and kinetics in real time (TaqMan, SDS 7700, ABI, Perkin Elmer).
  • Accessibility studies for DNA and RNA target sequences using FRET.
  • Fluorescence polarization protocols for monitoring ribozyme reactions (POLARstar, BMG, GmbH) and for DNA and RNA sequence detection.
  • Sequence detection with Syber green dye in real time quantitative PCR by Light Cycler (Roche Diagnostics, USA).
  • Single nucleotide polymorphism detection in real time with LightCycler hybridization probes (Roche Diagnostics, USA).

Gene Detection Technology: Research and Clinical Applications

  • Preparation of radio-labeled & fluorescent labeled RNAs (ribozymes and target substrates).
  • In vitro transcription of RNA.
  • Expression of ribozymes in yeast.
  • Isolation and purification of cellular RNA.
  • RNase Protection Assay.
  • Kinetic characterization of ribozymes & binding kinetics using fluorescence methods.
  • Designing, synthesis and characterization of allosteric ribozymes induced by small drug ligands (such as theophylline & caffeine).
  • Developed in utero microinjection techniques to transplant the bone marrow and stem cells to cure blood related genetic disease.
  • Harvest fetal liver, bone marrow and mouse embryonic stem cells for transplantation.
  • Culture mouse embryonic stem cell and in vitro differentiation into the blood cells.
  • Fractionation of cells using flow cytometry techniques.

Standard molecular biology techniques

  • Standard and site directed mutagenesis polymerase chain reaction (PCR).
  • Preparation and purification of plasmids.
  • Transformations and Transfection of DNA.
  • Cloning of DNA.
  • Solid phase synthesis of DNA (Gene Assembler, Pharmacia).
  • DNA sequencing & fragment analyses (ABI 310 Gene Sequencer, Perkin Elmer).
  • Quantitation of DNA, RNA and proteins.
  • Mammalian cell culture and yeast culture.
  • Gel electrophoresis (polyacrylamide and agarose).
  • Capillary gel electrophoresis (ABI 310 Gene Sequencer, Perkin Elmer).
  • Column/ gel / thin layer chromatography.
  • Autoradiography by phosphorimager (Storm, Molecular Dynamics, USA).
  • High Performance Liquid Chromatography (HPLC).
  • Preparation and purification of chemical reagents & solvents.
  • Enzyme/ protein/ purification and characterization.
  • Isolation of Genomic DNA, Genomic Library Construction.
  • Radioimmunoassay.

General molecular and biochemical techniques

mRNA preparation and purification, Primer designing, Real-time PCR, RT-PCR, DNA cloning, DNA sequencing, Isolation of Genomic DNA, Genomic library Construction, Transformation, Transfection, Cell culture, Plasmid purification, RNA probe making, Different kinds of microscopy, In situ hybridization, Southern blotting, Northern blotting, Western blotting, Spectrophotometery, In uteromicroinjection, Column chromatography, HPLC, PAGE, Agarose gel-electrophoresis, Enzyme assay, Protein assay, Enzyme/ Protein/ DNA purification, Histology, Phage display for tissue targeting, Radio-immunoassay.

Autologous stem cell transplant techniques

Developed the techniques to isolate the autologous stem cell from patient’s peripheral blood, bone marrow or fat tissues, characterize and activate those stem cells so they can be effectively used for the treatment of specific diseases.

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