27 de febrero de 2009

Buenas! En este mes nos interesaba llevar a cabo la digestion del pVinc con MspA1I y los minigenomas positivo y negativo con PmeI. Luego de la digestion esperamos ligar ambos plasmidos para obtener las construccion deseada y asi poder medir la replicacion de LuIII. En este mes pudimos extraer los fragmentos deseados y disenamos estrategias para la ligacion de ambos plasmidos. En este diseno, tenemos que anticipar los posibles obstaculos que pudiesen la ligacion. Entre ellos tenemos que determinar la concentracion apropiada para la ligacion. Una ligacion cuyo corte es cercano a los terminales del virus podria ser perjudicial, ya que podria formarse invaginaciones del terminal que pueden bloquear la ligacion. El progreso en la investigacion ha sido bastante bueno, pero nos pone nervioso que podria pasar con la ligacion.

Abstract del Bio-Minds Poster Day

Study of replication ratio of parvovirus LuIII minigenomes in Hela cells

Franklyn Rocha, Idaris de Jesus-Maldonado, Nanette Diffoot
Department of  Biology-University of Puerto Rico Mayaguez

LuIII is an autonomous icosahedral parvovirus that contains a single stranded DNA genome and infects eukaryotic cell lines. It has been proposed that an AT-rich region located near the right termini and unique in LuIII could be an element responsible for its encapsidation pattern. Even though our lab has found that AT-rich region is important for LuIII replication, we need some way to quantify the efficiency of replication before we can study the packaging of its DNA. Given the cis-acting sequences required for LuIII DNA excision and initiation of replication reside in the left and right termini, two minigenomes containing either termini (3′LuIII-GFP5′) but one of the them lacking the AT rich sequence (3′LuIIIAT(-)GFP5′) were constructed. These minigenomes also contains the GFP gene to visualize its replication efficiency. Hela cells were transfected and will be observed in fluorescence microscope. A flow cytometer will be used to determine the differences in fluorescent intensities. The resulting evidence may provide clues of the importance of AT-rich region in LuIII replication and its encapsidation. Understanding the mechanisms that mediate LuIII replication can help generate effective treatments against abnormal cell growth, such as those seen in cancerous cells.