Source: LOUISIANA STATE UNIVERSITY submitted to
A SYSTEM FOR RETROVIRUS-MEDIATED GENE DELIVERY INTO DOMESTIC FOWL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0190563
Grant No.
(N/A)
Project No.
LAB93525
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Aug 1, 2001
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Project Director
Ingram, D. R.
Recipient Organization
LOUISIANA STATE UNIVERSITY
(N/A)
BATON ROUGE,LA 70893
Performing Department
ANIMAL SCIENCE
Non Technical Summary
Conventional breeding programs in poultry are limited in the number of genes and alleles available in the poultry population. Genetic alteration of poultry may be useful in the identification of other genes that provide beneficial changes in production performance.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3033210104010%
3033210108040%
3033220104010%
3033220108040%
Goals / Objectives
Objectives: 1. To engineer a 3-component retroviral expression vector from a well characterized 2-component system that will allow for gene delivery into the avian embryo. 2. To develop a method for microinjecting infectious retroviral particles into early stage (day 1.5-2 incubation) chicken embryos to generate transgenic birds. 3. To characterize the expression of transferred marker genes, including spatial localization and temporal regulation.
Project Methods
Procedure: Construction of the three-component vector will utilize standard molecular biology techniques. Gag and Pol will be sub-cloned into an avian expression vector similar to Env. The psi packaging that mediates packaging of viral RNA into virion capsids has been defined for Rous-Sarcoma virus (RSV) (Knight et al, 1994). This domain will be cloned and a packaging vector will be constructed to allow the construct of interest to be packaged efficiently into infectious particles, as has been done previously for several mouse retroviral vectors. While the three-component vector is being constructed, the microinjection system and hatchery and grow out procedures will be established. The initial vectors to be used are replication-incompetent RSV vectors that express alkaline phosphatase, thus allowing for easy identification of infected cells in a tissue. Plasmids encoding viral genes and the marker gene will be transfected into 293T cells (ATCC), a human embryonic kidney cell line transformed with adenovirus and carrying a temperature sensitive T antigen. Cells will be washed and viral supernatants will be harvested every 24 h for 4 days. The infectious viral particles accumulate in cell culture supernatants, where they are concentrated by centrifugation for use in embryo injections. DF1 cells (ATCC), a chicken fibroblast cell line, will be used to determine viral titer. Injection of developing embryos with viral particles is relatively straightforward (Logan and Tabin, 1998). Fertile broiler eggs will be incubated for 48 h. A patch of shell approximately 2x2 cm, will be removed under aseptic conditions and the egg will be mounted in a micromanipulator in a sterile laminar flow hood. Using a low power dissection microscope, approximately 0.5-1 microliters of concentrated virus will be injected into the gut of the embryo (#25 in Fig. 1). The egg will be sealed with tape and returned to the incubator. This procedure has proved quite efficient in producing chimeric birds (Logan and Tabin, 1998; Chen et al., 1999). At hatch, the bird should be a mosaic for the gene of interest. Immunohistochemical staining for the alkaline phosphatase marker gene uses standard histologic techniques. The embryo is dissected, fixed, and a commercially available solution applied that yields a purple color when acted upon by alkaline phosphatase. This will allow us to assess how efficiently the vector is at delivery of genes into the embryo (Current Protocols, 1998; Marx et al., 1999; Skynner et al., 1999). Once the vector system has been optimized for gene expression, we can begin to investigate genes that may have commercial relevance for the improvement of poultry.

Progress 08/01/01 to 09/30/04

Outputs
Due to the initial low livability of embryos and thus low hatchability of eggs that were prepared to receive our proposed vector, numerous experiments were conducted to optimize livability and subsequent hatchability of windowed eggs or eggs which have had a small area of shell removed and resealed. Initial experiments studied the amount of egg albumen that could be removed without significantly affecting hatchability. When more than 1 mL of albumen was removed, hatchability as well as chick weight was significantly decreased regardless of egg size. Experiments were conducted to determine a sealing material that would result in the highest hatchability. Tested materials included several tapes, papafilm, cellophane and pieces of surrogate shell. Transparent adhesive tape produced the best results. In addition, egg orientation and turning was studied. The significantly greatest hatchability was obtained when eggs were incubated on their side with the window up and turned once per hour through 18 days of incubation. Other experiments tested the possible benefit of increased relative humidity during the incubation process. No benefits were observed when percent relative humidity was increased above the normally used 60% level. Additional experiments investigated the possible effects of egg storage on the hatchability of windowed eggs. When eggs were stored less that one week prior to undergoing the windowing procedure, there was no significant difference in hatchability. An experiment was also performed to determine if there might be an influence of egg shell color on the hatchability of embyros from windowed eggs. No difference was observed in hatchability when brown and white shelled eggs were compared.

Impacts
This research will help provide the necessary tools to make attempts at transgenics in poultry more successful. The resulting impact could provide for a more efficient and disease resistant bird.

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
In order for our proposed procedures to have maximum effectiveness, proper egg management during egg incubation must be determined. Thus experiments were conducted to determine the optimal incubator orientation for eggs which have had a small area of shell removed on their side and resealed. These eggs are normally referred to as windowed eggs. Hatchability was compared for windowed eggs set for the normal position, large end up, versus on their side with the window up for either one, two or three weeks of incubation. The significantly highest hatchability was obtained by incubating windowed eggs on their side for the entire three week incubation period. The benefits of turning windowed eggs during incubation were also evaluated in a series of trials. Windowed eggs incubated on their side were turned once per hour for either nine or eighteen days of incubation. The results indicated that hatchability was significantly improved by turning these eggs through day eighteen of incubation. Several experiments were conducted to determine the optimal material to apply as a seal over the window of eggs. Tested materials included: transparent adhesive tape, liquid skin, duct tape, masking tape, laboratory tape, parafilm, cellophane and surrogate shell. All non-adhesive treatments were held in place with egg albumen. Results indicated that all the different types of tape were superior to the other methods of sealing the window. Of the tapes, the scotch tape treatment appeared to be the most effective.

Impacts
This research will help provide the necessary tools to make attempts at transgenics in poultry more successful. The resulting impact could provide for a more efficient and disease resistant bird.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
Transgenics in poultry usually requires some type of egg albumen manipulation. Albumen must be removed so that the embryo can be positioned for transgenics. However, albumen is crucial to the proper development of the embryo. Thus, initial work on this project has focused on maximizing hatchability of eggs which have had some portion of their albumen removed. Experiments were conducted to determine the maximum amount of albumen which could be removed without affecting hatchability in various sized eggs. Albumen was removed in amounts ranging from 0.5-5.0 mL from eggs weighing 45-65g. Regardless of egg size when more than 1 mL of albumen was removed, hatchability as well as weight was significantly decreased. It has been concluded that a maximum of 1mL of albumen may be removed during this procedure. A current trial is being conducted to investigate egg shell sealing techniques and their effects on hatchability.

Impacts
Determining the maximum amount of egg albumen which may be removed from an egg without affecting hatchability will increase the success rate of transgenics in poultry.

Publications

  • No publications reported this period


Progress 01/01/01 to 12/31/01

Outputs
Work has recently begun on the project. Initial work has concentrated on maximizing and refining the embryo injection technique. In order to efficiently inject the embryo, some albumen must be removed in order to access the embryo freely. Currently, a trial is underway to investigate the maximum volume of albumen which can be removed without significantly affecting hatchability and chick weight. We are also developing procedures for sealing and incubating the injected eggs that will maximize hatchability. In addition, personnel are being trained to conduct these procedures.

Impacts
When completed this work will allow for a more efficient introduction of genes into the poultry genome.

Publications

  • No publications reported this period