1. Trends in Biotechnology 150513 TB 14 Microinjection, stem cell transfer, gene targeting, and use of retroviruses 1

2. Microinjection and use of retroviruses Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. 2

3. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x (though sometimes it is performed using a dissecting stereo microscope at 40-50x or a traditional compound upright microscope at similar power to an inverted model). 3

4. For processes such as cellular or pronuclear injection the target cell is positioned under the microscope and two micromanipulators are used to penetrate the cell membrane and/or the nuclear envelope. In this way the process can be used to introduce a vector into a single cell. 4

5. Microinjection can also be used in the cloning of organisms, in the study of cell biology and viruses, and for treating male subfertility through intracytoplasmic sperm injection 5

6. The use of microinjection as a biological procedure began in the early twentieth century, though even through the 1970s it was not commonly used. By the 1990s, however, its use had escalated significantly and it is now considered a common laboratory technique, along with vesicle fusion, electroporation, chemical transfection, and viral transduction, for introducing a small amount of a substance into a small target. 6

7. Pronuclear injection Diagram of the intracytoplasmic sperm injection of a human egg. Micromanipulator on the left holds egg in position while microinjector on the right delivers a single sperm cell. 7

8. Pronuclear injection is a technique used to create transgenic organisms by injecting genetic material into the nucleus of a fertilized oocyte. This technique is commonly used to study the role of genes using mouse animal models. 8

9. Pronuclear injection in mice The pronuclear injection of mouse sperm is one of the two most common methods for producing transgenic animals (along with the genetic engineering of embryonic stem cells). In order for pronuclear injection to be successful, the genetic material (typically linear DNA) must be injected while the genetic material from the oocyte and sperm are separate (i.e., the pronuclear phase). 9

10. Retroviruses http://www.saylor.org/content/BIO_Kimball/users.rcn.com/jkimball.ma.ult ranet/BiologyPages/R/Retroviruses.html#reverse The genome of retroviruses consists of RNA not DNA. HIV-1 and HIV-2, the agents that cause AIDS, are retroviruses. 10

11. A typical retrovirus is made of: an outer envelope which was derived from the plasma membrane of its host many copies of an envelope protein embedded in the lipid bilayer of its envelope a capsid; a protein shell containing two molecules of RNA and molecules of the enzyme reverse transcriptase 11

12. 12

13. Reverse transcriptase is a DNA polymerase that uses RNA as its template. It is able to make genetic information flow in the reverse (RNA ->DNA) of its normal direction (DNA -> RNA). 13

14. Infection of a host cell requires that the cell have a surface protein that can serve as a receptor for the envelope protein of the retrovirus. The envelope protein of HIV-1 binds to – CD4 molecules. It is this property that enables the virus to invade CD4 + T cells (and certain other cells that express CD4). – CCR5 (CC chemokine Receptor 5) — found on Th1 cells and macrophages. 14

15. All the proteins in the virus particle are encoded by its own genes. 15

16. When a retrovirus infects a cell its molecules of reverse transcriptase are carried into the cell attached to the viral RNA molecules. The reverse transcriptase synthesizes DNA copies of the RNA. These enter the nucleus and are inserted into the DNA of the host. 16

17. These inserts are transcribed by the host's enzymes into fresh RNA molecules which re- enter the cytosol where – some are translated by host ribosomes the gag gene is translated into molecules of the capsid protein the pol gene is transcribed into molecules of reverse transcriptase the env gene is translated into molecules of the envelope protein – other RNA molecules become incorporated into fresh virus particles 17

18. 18

19. The genome of retroviruses is flanked at each end by repeated sequences ("R") that enable the DNA copy of the genome to be inserted into the DNA of the host and act as enhancers, causing the host nucleus to transcribe the DNA copies of the retroviral genome at a rapid rate. 19

20. The retroviral genome also contains a packaging signal sequence ("P") which is needed for the newly-synthesized RNA molecules to be incorporated in fresh virus particles. 20

21. Most retroviruses also contain one or more additional genes. Some of these represent RNA copies of genes that earlier were picked up from their eukaryotic host. Several cancers in animals are caused by retroviruses that have, at some earlier time, picked up a proto-oncogene from their mammalian host and converted it into an oncogene. 21

22. Animation of Virus as a Vector http://www.iupui.edu/~wellsctr/MMIA/htm/a nimations.htm 22

23. 23