scientificprotocols

Authors: Subeer S. Majumdar , Suveera Dhup & Abu-ul Usmani 

Abstract

Presently used techniques for making transgenic animals are cumbersome; require trained man-power, costly infrastructure and large number of zygotes at the expense of several females. The ability of the male germ cells to integrate foreign genes provides opportunity for developing alternate methods for generation of transgenic animals. We have developed a reproducible protocol for transfecting mammalian and non-mammalian genes in repopulating undifferentiated spermatogonial cells through in vivo electroporation of the testis. More than 90% of the male mice electroporated with transgene of choice sire transgenic pups upon mating with the wild type (WT) females. Such males serve as permanent resource for the production of transgenic founders. This technique has several advantages over the presently used techniques including drastic reduction in the time required and in the usage of animals for generation of transgenic progeny. Henc

scientificprotocols

Authors: Bruce E. Tabashnik , David W. Crowder & Yves Carrière 

Introduction

Genetically modified crops producing insecticidal proteins from Bacillus thuringiensis (Bt) for insect control have been planted on more than 200 million ha worldwide since 1996 [1]. Evolution of resistance by insect pests threatens the continued success of Bt crops [2, 3]. To delay pest resistance, refuges of non-Bt crops are planted near Bt crops to allow survival of susceptible pests [4, 5]. We used computer simulations of a population genetic model to determine if predictions from the theory underlying the refuge strategy match outcomes in the field documented with monitoring data [6]. The computer program is called SERBt for Simulated Evolution of Resistance to Bt crops. For the six major pests modeled, the simulation results corresponded with the field data. In particular, the simulations indicated that resistance would evolve fastest in Helicoverpa zea, a major cotton pest in the U.S., and this insect was the fi

scientificprotocols

Authors: Paulina Carriba , Gemma Navarro , Francisco Ciruela , Sergi Ferre , Vicent Casadó , Antonio Cortes , Josefa Mallol , Enric Canela , Carmen Lluis & Rafael Franco 

Introduction

Combining BRET and FRET in the Sequential BRET-FRET (SRET) new technique permits heteromers formed by three different proteins to be identified. In SRET experiments, the oxidation of a Rluc substrate triggers acceptor excitation by BRET and subsequent energy transfer to a FRET acceptor. Thus, SRET requires the co-expression of three fusion proteins, one coupled to Rluc, another conjugated with GFP2 or YFP, and the third with YFP or DsRed. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will enable us to better understand how signals are integrated at the molecular level.

Reagents

1. cDNA for functionally validated fusion proteins in suitable expression vectors.

• Suitable cells for transfection (e.g., HEK293)

scientificprotocols

Authors: Sergé Arnauld , Bertaux Nicolas , Rigneault Hervé & Marguet Didier 

Introduction

Single fluorescence molecule microscopy provides a privileged approach achieving a spatial resolution close to the molecular scale (1-5). The position of an isolated fluorescent molecule is accurately determined by the center of the signal. The counterpart is a necessity for imaging sparse fluorescent molecules. Consequently, single particle tracking (SPT) is only amenable when handling a very limited number of targets at the same time.

This major drawback has been partly circumvented by the development of super-resolution optical imaging techniques (6). Among them, photoactivation localization microscopy (PALM (7), FPALM (8)) or stochastic optical reconstruction microscopy (STORM (9)) permit the localization of thousands of single fluorophores per acquisition. This is experimentally performed by sequentially turning on a fraction of the fluorescently-labeled molecules. The combination of PALM with live-cell

scientificprotocols

Authors: Suenori Chiku , Kimio Yoshimura & Teruhiko Yoshida 

Introduction

There are two kinds of applications of principal component analysis (PCA) to analyze population substructures of genetic polymorphism data. One application is for an individual covariance matrix, and the other application is for a marker covariance matrix. The former method is already implemented in EIGENSTRAT 1; the latter method, however, is not common because it cannot be applied, if data include missing typing data (allele call). Here, we describe some modification of a Mixture Model [2] (MM), so that it can handle data with missing allele calls (we call it a compensated mixture model (CMM) protocol). MM applies PCA to a marker covariance matrix before applying the normal-distribution mixture model.

Equipment

1. Genotype data file on markers (e.g. SNPs in our GWAS on gastric cancer), which were selected so that the marker loci would be independent each other (an example of such selection criteria is given below for

scientificprotocols

Authors: Young Kwon , Hye-Seok Shim , Xiaoyue Wang & Craig Montell 

Introduction

When given a choice between two temperatures, Drosophila larvae will select the preferred temperature. This protocol outlines a step-by-step procedure for performing a two-way choice test on Drosophila larvae, to identify the preferred temperature.

Procedure

Rearing 3rd instar larvae

scientificprotocols

Authors: Gargioli Cesare 

Introduction

With this technique it is possible to label the blood vessel in vivo in order to analyze in whole mount the quantity and quality of blood vessel.

Reagents

1. 1.0 ml syringe

• China Black Ink

scientificprotocols

Authors: Wei Guo & Hong Wu 

Introduction

Rosa26-LacZ transgenic reporter mice are a very common reporter line to determine Cre activity (1). Since the disruption of target gene(s) and the expression of the Rosa26-LacZ transgene are both controlled by the same Cre recombinase, LacZ expression/activity can serve as a useful marker of the cells with targeted gene disruption. X-gal staining is a common assay used to detect LacZ+ cells in many tissues. In 1988, Nolan, et. al. (2) developed FACS-Gal analysis, a FACS-based detection of LacZ+ cells with a fluorogenic substrate, FDG, of galactosidase (LacZ enzyme). Galactosidase cleaves FDG and releases a fluorescence product FITC (3). However, it is common that LacZ signals varies from sample to sample. Here we establish a procedure to ensure reliable and consistent results for the whole hematopoietic system, with the exception of terminally differentiated erythroid cells, or red blood cells that express much lower LacZ proteins.

Reagents

1. 70µm c

scientificprotocols

Authors: Wei Guo & Hong Wu 

Introduction

Genomic instabilities including chromosomal translocations are frequently associated with genetic diseases and cancer, especially leukemia. Cytogenetic studies of these diseases requiring preparation of metaphase chromosomes are often key to revealing their chromosomal abnormalities. Treatment of in vitro disease cell cultures with cell cycle inhibitors, e.g. colcemid, have proved to be a very simple and effective metaphase preparation method for cytogenetic studies. Two good examples are metaphase preparation from cultured cells and some types of myeloid leukemia. However, this approach has some limitions and disadvantages: 1) it is difficult to grow many types of primary cell in vitro; 2) in vitro cultures tend to introduce additional artificial mutations to the genome. To overcome these issues, we have established an approach to prepare metaphase chromosomes directly from murine bone marrow. With this new approach, we were able to get 5-10% of metaphase c

scientificprotocols

Authors: Wei Guo & Hong Wu 

Introduction

Human leukemia has been determined and classified with the help of flow cytometry for the past two decades. Past attempts to detect leukemia blasts relied on both forward and side scatter (FSC and SSC) based on cell size and granularity. However, this technique failed to show a clean separation of blasts from normal lineage cells. In 1993, Borowitz, et al developed flow cytometric analysis to distinguish human leukemia blasts from other normal lineage cells by using fluorescence-conjugated CD45 antibodies (1). On CD45-SSC plots, the blasts are distinctly located below granuocytes in human acute myeloid leukemia. This technical advance has significantly improved diagnosis and classification of human leukemia.

Animal models mimicking human leukemia help us make a deeper understanding of leukemia progression and develop more effective therapeutic interventions in human leukemia. Recently, two groups of researchers published Bethesda proposals for classificatio