CLONING & EXPRESSION OF TCL

The Cloning and Expression of TCL via Baculovirus in Insect Cells

The Cloning and Expression of TCL via Baculovirus in Insect Cells

Introduction

FGD6 regulates the Rho protein signal transduction. It May activate CDC42, a member of the Ras-like family of Rho- and Rac proteins, by exchanging bound GDP for free GTP. May play a role in regulating the actin cytoskeleton and cell shape (By similarity). Emigration of TCL from blood into sites of inflammation and immune responses is essential for host defense mechanisms. Local tissue irritation causes TCL to stick to blood vessels, to pass through them, and finally to accumulate at irritated sites. The known insect cells are divided into two major subfamilies based on the spacing of the first two cysteines in the conserved motif and the chromosomal localization of their genes (Fangerau, 2005).

Ligation process

It is the process of annealing or joining, such as the DNA ligase-catalyzed joining of two DNA fragments to form a recombinant molecule.

Preparation of DNA for ligation

Prior to ligation, compatible insert and vector DNA must be prepared. Both need compatible termini and must be free of contaminating chemicals such as phenol, ethylenediaminetetraacetic acid (EDTA) or ethanol that may inhibit ligation. Vector DNA sis typically prepared by digestion with the relevant restriction enzyme, then dephosphorylated by incubation with alkaline phosphatase to remove the terminal 5' phosphate groups and so prevent vector self-ligation.

Ligation reaction

PCR is an extremely powerful technique as it allows for minute quantities of DNA (or messenger ribonucleic acid (mRNA)) to be amplified up to cloneable quantities. PCR products synthesized using enzymes such as Taq DNA polymerase (that lack 3'-5' exonuclease activity) have a single adenosine 3' overhang and those synthesized with 'proofreading' polymerases which possess 3'-5' exonuclease activity are generated with blunt ends (Jensen, 2008).

Bacterial transformation

M13 is a filamentous bacteriophage, with a 6.4 kb single-stranded genome, that infects E. coli. An infected cell produces 100-200 double-stranded (or replicative form (RF)) copies of the phage genome.

These encode genes that coordinate packaging of one of the phage genome DNA strands into a proteinaceous viral coat and their extrusion from the cell where they are then free to infect further cells. A series of M13-based cloning vectors have been developed (Messing, 1983), and found capable of accepting inserts several times larger than the M13 genome.

Miniprep Method

The technical report below by Chowdhury, K. (1991) is a very fast, simple and one step 'miniprep' procedure. The quality and quantity of DNA obtained by using this procedure is similar to those obtained by the other commonly used procedures of Serghini et al. (1) or Birboim and Doly.

Electrophoresis

It is the movement of charged colloidal particles through the medium in which they are dispersed as a result of changes in electrical potential. Electrophoretic methods are useful in the analysis of protein mixtures because protein particles move with different velocities depending principally on the number of charges carried by the particle.

PCR -electrophoresis

When typing is based on the REP sequence (= REP-PCR; ...