Animal biotechnology lecture 2

1. Animal biotechnology lecture 2 Dr. Ziad Jaradat

2. Animal Biotechnology & Transgenic Animals • Since the early 1980s, fruit flies, fish, sea urchins, frogs, laboratory mice and farm animals, such as cows, pigs, and sheep have been successfully produced. • The ability to manipulate the genome of the whole animal and the production of transgenic animals has influenced the science dramatically in the last 15 years. • The procedure for introducing exogenous donor DNA into a recipient cell is called Transfection. • Chromosomes are taken up inefficiently so that intact chromosomes rarely survived the procedure. Instead the recipient cell usually get a part of the DNA.

3. Now, with the advent of the recombinant DNA, the possibility of introducing a particular segment of DNA become possible. However, still there are always some problems of the stability of the new inserts (transient transfectants). • An exciting development of transfection techniques is the application of DNA technology to introduce genes into animals.

4. An animal that gains new genetic information from the addition of foreign DNA is described as Transgenic while the introduced DNA is called the transgene. • The transgenes are introduced into the pronuclei of fertilized eggs by injection, and the injected embryos are incubated in vitro or implanted into the uterus of a pseudopregnant female for subsequent development. What is Pronucleus? For a short time after fertilization, the male pronucleus and female pronucleus exist separately. • Female pronucleus; In the maturing of the ovum preparatory to impregnation, a part of the germinal vesicle becomes converted into a number of small vesicles, which aggregate themselves into a single clear nucleus which travels towards the center of the egg and is called the female pronucleus.

5. Male pronucleus; In impregnation, the spermatozon which enters the egg soon loses its tail, while the head forms a nucleus, called the male pronucleus, which gradually travels towards the female pronucleus and eventually fuses with it, forming the first segmentation nucleus. The male pronucleus is larger than the female’s and can be seen fairly easily under a light microscope.

7. Synopsis of the transgenesis process; • Plasmids carrying the gene of interest are injected into the germinal vesicle (nucleus) of the oocyte or into the pronucleus (before uniting with the gamete) of the fertilized egg. • The egg is implanted into a pseudopregnant mouse • After birth, the recipient mouse can be examined to see whether it has gained the foreign DNA and if so whether it is expressed. • As a result; multiple copies of transgenes are integrated at random locations in the genome of the transgenic individuals. • The transgenes in many transgenic individuals are also transmitted through the germline to subsequent generations.

8. Note; If the transgenes are linked with functional promoters, expression of transgenes as well as display of change in phenotype is expected in some of the transgenic individuals • Questions to be asked about any transgenic animal are; • how many copies it has of the foreign DNA (varies 1-50)

9. where these copies are located [usually multiple copies are integrated into a tandem array (arranged adjacent to each other) into a single chromosomal site] • whether they are present in the germ line and inherited in Mendelian manner. • can the gene be expressed independently? i.e does the regulatory elements function independently • are transfected genes expressed with the proper developmental specificity? • A good result if we obtain 15% of the animals to be transgenic. • In the progeny of the infected animal, the expression of the donor gene is extremely variable and that could be dependent on the place of integration of the new DNA.

10. Transgenesis; Methodology • Transgenic technology has been developed and perfected in the laboratory mouse. Since the early1980’s hundreds of different genes have been introduced into various mouse strains. These studies have contributed to; • understanding of gene regulation • tumor development, example introducing oncogenes and observe the effect

11. immunological specificity, example producing knockout genes that are responsible for some immunological aspects • molecular genetics of development • other biological interests such as examining the possibility of using transgenic animals in the industrial production of human therapeutic drugs.. etc.

12. Methods of gene transfer in animals For transgenesis, DNA can be introduced into mice by one of the following methods; • Retroviral vectors that infects the cells of an early stage embryo prior to implantation into a receptive female. • Microinjection into the enlarged sperm nucleus (the male pronucleus) of a fertilized egg • Introduction of genetically engineered embryonic stem cells into an early stage developing embryo prior to implantation into a receptive female. • Transfer of diploid somatic nuclei into an enucleated oocyte.

13. Retrovirus-Mediated Gene Transfer • The most useful vectors for the purpose of gene isolation are those that lend themselves to the production of libraries consisting of overlapping fragments of genomic DNA, ideally encompassing the entire genome several times. • Exmaple; bacteriophage λ genomic library of 106 viruses each containing on average 20 Kb of DNA, represents 6-7 copies of the entire mouse genome and the probability that each gene is represented is very high. • Retroviruses can be used for the transfer of foreign genes into animal genomes.

14. This can best be done at 4-16 cell stage embryos. However, it can be done up to midgestation, but with incomplete infections i.e low infectivity rate. • Immediately following infection, the retrovirus produces a DNA copy of its RNA genome using its reverse transcriptase. • Completion of this process requires that the host cell undergoes the S phase of the cell cycle. Therefore, retroviruses effectively transduce only mitotically active cells. • Modifications to the retrovirus frequently consist of removal of structural genes, such as gag, pol, and env, which support viral particle formation.

15. Additionally, most retroviruses and complementary lines are ecotropic in that they infect only rodents, such as rats and mice, and rodent cell lines rather than humans. • The DNA copy of the viral genome, or provirus, integrates randomly into the host cell genome, usually without deletions or rearrangements. • Because integration is not by way of homologous recombination, this method is not used effectively for site-directed mutagenesis. • Very high rates of gene transfer are achieved with the use of retroviruses.

16. Table of common vectors used for such purpose Vector Origin Insert size rangeMulticopy plasmids multicopy plasmids up to 20 kb • Lambda vectors Bacteriophage λ up to 30 kb • Cosmid Bacteriophage λ up to 40 kb • P1 artificial chrom Bacteriophage P1 80-90 kb • Bacterial artificial chrom. Large Bacteria plasmid 100-300 kb (F factor) • Yeast chrom. (YAC) Yeast chromosome 100-1000 + kb + means indefinite.

17. Disadvantages of this method include: • Low copy number integration. • Additional steps required to produce retroviruses. • Limitations on the size of the foreign DNA insert (usually 9 to 15 kb) transferred. • Potential for undesired genetic recombination that may alter the retrovirus. • High frequency of mosaicism. • Possible interference by integrated retroviral sequences in transgene expression.

18. The genome of the retroviral strain can be integrated into the same nucleus as the transgene. This means that the virus itself could be produced by the transgenic organism and create a problem especially if the animal will be used for production of food. • Also the provirus attracts methylation which possibly in conjugation with other mechanisms disables its expression when it passes through the germ line. • Due to this, and to the availability of other alternative methods, the retroviral vector method is rarely used for producing transgenic animals that have a commercial potential.

19. DNA Microinjection Method Because of the disadvantages of the retroviral vectors, microinjection of DNA is currently the preferred method for producing transgenic mice. • First - you need the gene of interest in the proper form. Alinear transgene construct is made, which contains: • the structural gene of interest, with introns • a strong mouse gene promoter and enhancerto allow the gene to be expressed • vector DNA to enable the transgene to be inserted into host DNA • The immature female mice will be induced to superovulate by sequential administration of FSH/LH and HCG and mated to fertile males. One-celled embryos are flushed from the oviducts and placed in a drop of medium and viewed by phase-contrast or interference microscopy.

20. This procedure has the following steps; • The number of available fertilized eggs that are to be inoculated are increased by stimulating donor females to superovulate. • This can be done by • Giving the mice an initial injection of pregnant mare’s ( an adult female of horse or related mammal) serum • Another injection about 48 hours later of human chorionic gonadotropin (hCG). By this protocol the female produces about 35 eggs instead of the normal number of 5-10.

21. These females are mated, then sacrificed and the fertilized eggs (oocytes) are flushed from their oviducts and recovered. • Eggs are treated with hyaluronidase to remove adherent follicle cells. • Unfertilized eggs are discarded • The eggs are inoculated immediately with the transgene, briefly; • embryo at the pronuclear stage is held in place by suction. • a micro needle loaded with a suspension of plasmid DNA will be prepared. • It is introduced through the zona pellucida and plasma membrane into the most accessible pronucleus (usually the male) and • several hundred molecules of the recombinant DNA are injected in a volume of approximately 1 picoliter (p1). • on a good day several hundred eggs can be injected. • The male pronucleus can be located by using dissecting microscope and the eggs then can be maneuvered, oriented and held in place while the DNA is microinjected.

22. oocyte Pippet Micro needle

24. After inoculation, 25-40 eggs are implanted microscopically into a foster mother who has been made pseudo-pregnant by being mated to a vasectomized male so that none of the eggs of the foster mother will be fertile therefore, the foster mother will deliver pups from the implanted fertile eggs three weeks after the inoculation. • After birth, the presence of foreign material is studied by DNA hybridization with appropriate probes or PCR. • A transgenic mouse can be mated to another to produce transgenic homozygous transgenicanimal.

25. More details on gene transfer.

26. Gene Transfer to Animal cells Dr. Ziad Jaradat

27. There are four major strategies for gene transfer to animal cells, two of which are considered biological mechanisms using virus (transduction) or bacterial that invade animal cells (bactofection), those methods involve infection while the other two are chemical and physical methods which do not involve infection thus termed transfection. • Virus (transduction); the transferred gene represents part of the viral genome • Bacteria (bactofection); the gene will be transferred as a plasmid

28. Chemical transfection • ; DNA will be taken from the surroundings when the DNA is presented as a synthetic complex either as; • a complex with overall positive charge, allowing it to interact with negatively charge cell membrane and promote uptake by endocytosis • as lipophilic complex that fuses with the cell membrane and deposits the transgene directly into the cytoplasm.

29. Physical transfection; • in this method, naked DNA is deposited directly into the cell by exploiting a physical force. This includes; • microinjection • particle bombardment • ultrasound • electroporation • Which ever method used, the result is called transformation which is a change in the recipient cell’s genome caused by the acquired transgene.

30. Chemical Transfection Techniques • Calcium phosphate method; involves the formation of a fine DNA/calcium phosphate co-precipitate which first settles on the cells and then internalized by endocytosis. • The precipitate must be formed freshly at the time of transfection. The DNA escapes and reaches the nucleus and can be then expressed. Since the cells must be coated by the calcium complex, monolayers of cells must be used for maximum efficiency. However, this method gives only 1-2% transfection efficiency.

31. Transfection with polyplexes; this is more efficient than that with calcium as it gives more uniform particle size. Polyplexes are a polycationic compounds that form soluble complexes (polyplexes) through spontaneous electrostatic interaction with DNA. • This method is adapted as a plasmid DNA transfer. The efficiency of the method can be increased by exposing cells to osmotic shock or treatment of cells with chloroquine. • Anew generation of the polycationic compounds has been developed such as poly-L-lysine and synthetic poly amines, polyethelylenimines and dendrimers (highly complex molecules built in layers from a central initiator such as ammonia or ethylenediamine).

32. Transfection with liposomes and lipoplexes • This can be done by packaging the DNA inside a fusogenic phospholipid vesicle which interacts with the target cell membrane and facilitate DNA uptake. • Briefly, bacterial cells will be transformed with suitable plasmid vector and treated with chloramphenicol to amplify plasmid copy number.

33. The cells will be treated with lysozyme to remove cell wall, resulting in protoplasts that will be centrifuged gently onto a monolayer of mammalian cells to promote fusion among them using polyethylene glycol. • Using lyposomes is more commonly used for this type of transfection. This method is commonly known as lipofection. This method is far more efficient than chemical transfection method. With this method up to 90% of cells in culture dish can be transected.

34. Physical transfection methods • Electroporation • Ultrasound • both methods create transient pores in the cell

35. Electroporation • is a physical transfection technique involves creating transient nano-meter size pores in the cell membrane by exposing cells to a brief pulse of electricity. The most critical parameter is the intensity and duration of electrical pulse. • Electroporation can be used for in vivo gene transfer particularly for surface or near surface tissue such as skin, muscle and certain tumors or even internal tissues such as liver. This can be achieved by direct application of electrodes to the skin following shaving and mild abrasion with the DNA being injected into the skin before electroporation.

36. Ultrasound transfection • Involves the exposure of cells to a rapidly oscillating probe such as the tip of sonicator. In this method the application of ultrasound waves to a dish or cells or a particular tissue results in the formation and collapse of bubbles in the liquid, including the cell membrane, a process known as cavitation. • The transient appearance of such cavities allows the DNA to cross the membrane into the cytoplasm. This method can be used both for in vivo or in vitro as the plasmid DNA is left structurally intact. As for electroporation, the DNA will be injected and then the ultrasound will be applied.

37. DNA microinjection Direct transfer of DNA into the cell without a carrier is called DNA microinjectin. This can only be done for only few cells at a time. This technique is used mainly for large cells such as oocytes, eggs and the cells of early embryos. The DNA can be directly injected into tissues, such as skin, muscle or internal organs or it can be injected into the blood.

38. The process is remarkably efficient. Up to 60- 66%% of the embryos survive injection and up to 25-30% of the embryos transferred to the oviduct survive to birth and about 25% of pups are transgenic (transgenic founders). Thus, from 1000 inoculated fertile eggs, 30-50 (3-5%) transgenic pups are produced. The injected DNA gets incorporated at random sites within the genome and often multiple copies are incorporated at one site, therefore, not all the transgenic animals will have the desired traits. 38

39. Particle bombardment • is another direct delivery method initially developed for the transformation of plants. This method involves coating small metal particles with DNA and accelerating them into target tissues using a powerful force such as the blast of high pressure gas or an electric discharge through a water droplet. In animals, this method is used for tissues such as skin cells in vivo rather than cultured cells.

40. Bacterial vectors for gene transfer The exploitation of living bacteria for gene transfer is central to the genetic manipulation of plants. Agrobacterium tumefaciens and its close relatives have been used for 20 years to generate transgenic plants. • Recently, A. tumefaciens has been used to transfer DNA to human cells. The protoplast fusion technique can be considered as highly efficient form of bactofection. However, this type needs human intervention, while the A. tumefaciens does not require human intervention.

41. How does the bacterial transfer of DNA happen? • The bacteria invades the host animal cells and undergo lysis within them releasing their plasmid DNA. Example of these bacteria including Salmonella species (lysis occurs in the phagocytic vesicle), Listeria monocytogenes and Shigella flexneri (lysis occurs for these two species after they escape from the vesicle).

42. The plasmid DNA then finds its way to the nucleus where it gets incorporated with the cell’s genome and gets expressed. • Contrary to the above mentioned bacteria, A. tumefaciens does not invade the cell, instead, it attaches itself to the outside surface followed by conjugation.

43. Viruses That are used as gene transfer vectors • Virus particles have a natural ability to adsorb to the surface of the cells and gain entry. This can be exploited to deliver recombinant DNA into animal cells. • Several classes of viruses has been used for gene therapy and at least 8 has been used in clinical trials. Transgenes may be incorporated into viral vectors either by addition to the whole genome or by replacing one or more viral genes. This can be done by ligation or by homologous recombination.

44. If the transgene replaces a none essential gene the vector is described as helper-independent • If it replaces an indispensable gene, then this vector will be helper dependent. • It is generally recommended to use vectors from which all viral coding sequences has been deleted such vectors are described as fully deleted or gutted or gutless vectors. These vectors contain just the cis-acting elements required for packaging and for genome replication.

45. Advantages of such vectors • high capacity for foreign DNA • because no viral gene products are made, the vector has no intrinsic cytotoxic effects. Adeno virus • These are viruses with a linear, double stranded genome, of approximately 36 kb. These are used frequently due to certain advantages; • stability • high capacity for foreign DNA • wide host range including none-dividing cells • Ability to produce high titer stocks up to 1011 pfu/ml.

46. Adeno-associate virus (AAV) • These viruses are not genetically related to adenovirus but is so-called because it was first discovered as a contaminant in an adenovirus isolate. • The AAV is a single stranded DNA and is a member of the parvovirus family. • It is naturally replicating deficient, thus it requires the presence of another virus such as adenovirus to complete its infection cycle. AAV replicates lytically and produces thousands of progeny virions.

47. The dependence of AAV on a heterologous helper virus such as adeno virus provides an unusual degree of control over vector replication making AAV one of the safest vectors to use for gene therapy. • Other advantages of this viral vector is the wide host range that it exhibits including none dividing cells.

48. The AAV genome is small (5 kb) and comprises a central region containing rep (replicase) and cap (capsid) genes flanked by 145 kb inverted terminal repeats. • Foreign DNA replaces the cap region and gets expressed by indigenous promoter. However rep proteins might interfere with the expression process thus responsible for some of the cytotoxic effects of the virus.

49. New vectors are designed with the deletion of the rep and cap genes and only utilizing the repetitive sequence which are the only elements required for replication, transcription and proviral integration. • AAV vectors have been used to introduce genes efficiently into many cells including liver, muscle, and neurons.

50. Baculovirus vectors (BV) • Baculovirus promote high level of transgene expression in insect cells but can also infect mammalian cells. • Have rode shape capsid and large double-stranded DNA genomes. They productively infect arthropods particularly insects. • BV vectors are used mainly for high–level transient protein expression in insects and insect cells.