Identifying Strategies to Clone Viral Genes into Yeast Expression Vectors

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Table of Contents

CHAPTER I: INTRODUCTION3

Background3

Introduction4

Vectors for yeast and other fungi5

Gene delivery systems: nonviral vectors14

Yeast Expression- Saccharomyces Cerevisiae16

Cloning Gag Pol19

Adeno Associated Virus in Yeast Expression22

PUC19 PLASMID, TOPO TA cloning, Likers and PCR24

Problem Statement25

Scope of the Study26

Aims and Objectives27

Purpose of the Study27

References28

Identifying Strategies to Clone Viral Genes into Yeast Expression Vectors

CHAPTER I: INTRODUCTION

Background

Viruses have evolved to become highly efficient at nucleic acid delivery to specific cell types while avoiding immunosurveillance by an infected host. These properties make viruses attractive gene-delivery vehicles, or vectors, for gene therapy. Several types of viruses, including retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus, have been modified in the laboratory for use in gene therapy applications. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits geneexpressionin vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many nondividing and dividing cell types, but has a limited DNA capacity. Alternatively, chimeric viral-vector systems that combine advantageous properties of two or more viral systems are also being explored. Although viral-mediated gene delivery has proved to be the most efficient means of gene transfer, nonviral means are also under development. Many of these nonviral systems incorporate portions of viralvectors to increase the efficiency of gene delivery or expression. Retrovirus, adenovirus, and AAV vectors are being evaluated currently in several Phase 1 clinical trials for treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, and arthritis.

A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H. polymorpha-derived integration sequence. The dimorphic yeastArxula adeninivorans, which is currently being assessed for heterologous geneexpression, was the main focus of the study. As in H. polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene. Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae. Based on these results the design of a 'universal' fungal vector appears to be feasible.

Introduction

Most cloning experiments are carried out with E. coli as the host, and the widest variety of cloning vectors are available for this organism. E. coli is particularly popular when the aim of the cloning experiment is to study the basic features of molecular biology such as gene structure and function. However, under some circumstances it may be desirable to use a different host for a gene cloning experiment. This is especially true in biotechnology, where the aim may not be to study a gene, but to use cloning to control or improve synthesis of an ...