Advances Of Molecular Genetics Of Poultry


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  • (1) Maxam-Gilbert 法: 以四種化學反應分別對四種鹼基作用,每一反應只對單一種鹼基進行修飾,而在該鹼基的地方斷開,得到一系列長度不同的核酸片段。 電泳可依照這些 DNA 片段的大小,在膠體中排開,即可依序判讀 DNA 分子上核 苷 酸的序列;比較如此四組鹼基序列電泳,即可組合成整段 DNA 。 (2) Sanger 法: 以樣本 DNA 為模板,使用 DNA polymerase 進行試管中 DNA 生合成。 四個反應中,每個反應各缺單一種核苷酸,而代以其 類似物 (analogue) ,則部分合成反應會停在該類似物的核 苷 酸處,造成各種長短不一的 DNA 片段,以電泳分離如上,即可組合判讀 DNA 的序列。 上述兩種方法,均以 32 P 標示在核酸分子上,以便顯像各不同長度的核酸片段。
  • 目前 都用 Sanger 的生合成法,以目標核酸為模板,加入四種核苷酸及 polymerase ,複製該段核酸;但除了所加的四種核苷酸外,額外加入其中任一種核 苷 酸的雙去氧衍生物 dideoxynucleotide ,則合成反應將可能止於這種核苷酸的位置上,因而可得到各種長短不同的片段,以電泳可以依序排列出來。
  • 質體 可以由一種宿主傳到另一種宿主菌,因此抗藥性也可以因此傳播開來,使得很多細菌很快對抗生素產生抗藥性。 遺傳工程也是利用質體的這種傳播功能,把一個被修改過的重組質體,放到宿主細菌中去表現,生產我們所要的物質。
  • Fig 22.13 The production of transgenic animals by microinjection of DNA into fertilized eggs.
  • Advances Of Molecular Genetics Of Poultry

    1. 1. Advances in molecular genetics of poultry Presented By : Alok Bharti MVSc. [ AGB ]
    2. 2. What is molecular genetics <ul><li>Is the study of genetic make up of individuals at the DNA level </li></ul><ul><li>The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. </li></ul>
    3. 3. Beginning of Molecular Biology <ul><li>The Modern of molecular biology begins in the 1930s </li></ul><ul><li>with the convergence of various, previously distinct biological disciplines: biochemistry, genetics, microbiology, and virology </li></ul><ul><li>With the hope of understanding life at its most fundamental level </li></ul>
    4. 4. Warren Weaver <ul><li>While molecular biology was established in the 1930s, the term was first coined by Warren Weaver in 1938 </li></ul>
    5. 5. Early Vision on Molecular Biology
    6. 6. Conventional methods <ul><li>Depends on phenotypic selection. This </li></ul><ul><ul><li>Narrows the genetic base of a population. i.e. Influenced by environmental selective pressure </li></ul></ul><ul><ul><li>Only be applied to traits that are easily measured </li></ul></ul><ul><ul><li>Takes long period of time </li></ul></ul><ul><ul><li>High costs </li></ul></ul><ul><li>Limited in </li></ul><ul><ul><li>sensitivity </li></ul></ul><ul><ul><li>specificity </li></ul></ul>
    7. 7. Advantages of molecular genetics <ul><li>Not affects by environmental effects. i.e. h 2 =1. </li></ul><ul><li>Can be available at an early age (in principle at the embryo stage) </li></ul><ul><ul><li>Allowing early selection </li></ul></ul><ul><ul><li>Generation interval reduction </li></ul></ul><ul><li>Especially beneficial for sex-limited traits, traits that are difficult to record, or traits that require slaughter of the animal (carcass traits) </li></ul>
    8. 8. Scope of molecular genetics <ul><li>Molecular analysis of genetic diversity </li></ul><ul><li>Animal identification and traceability </li></ul><ul><li>Reproductive enhancement </li></ul><ul><li>Transgenic livestock </li></ul><ul><li>Germ line manipulation </li></ul><ul><li>Gene based trait selection </li></ul><ul><li>Poultry health: diagnosis , protection and treatment </li></ul>
    9. 9. Molecular genetics tools Molecular Genetics Cloning Technology DNA Sequencing Technology Transgenesis Technology PCR Technologies Marker Assisted Selection
    10. 10. Introduction : Molecular Markers <ul><li>Also known as a  genetic marker . </li></ul><ul><li>It usually applies to an  allele ,  DNA marker , or cyto genetic marker </li></ul><ul><li>  used as an experimental  probe  to keep track of an individual, a  tissue , a  cell , a  nucleus , a  chromosome , or a  gene .  </li></ul>
    11. 11. Molecular markers are chosen because they are <ul><li>Relatively cheap </li></ul><ul><li>Amenable to High throughput protocols </li></ul><ul><li>Distributed over most of genome </li></ul><ul><li>Not dependent on high DNA sample availability </li></ul>
    12. 12. Why do we care about variations? underlie phenotypic differences allow tracking human/animal history (ancient and modern) cause inherited diseases
    13. 13. Molecular Marker Techniques <ul><li>Restriction Fragment Length Polymorphism (RFLP) </li></ul><ul><li>The technique centers around the digestion of genomic DNA digested with restriction enzymes [Forming recognition sites] </li></ul><ul><li>These recognition sites are not associated with any type of gene and are distributed randomly throughout the genome. </li></ul><ul><li>When genomic DNA is digested with one of these restriction enzymes, (of which there are thousands, each cutting at a specific sequence), a series of fragment are produced of varying length. </li></ul>
    14. 14. <ul><li>These fragments are separated using PAGE and yield a characteristic pattern </li></ul><ul><li>Variations in the characteristic pattern of a RFLP digest can be caused by </li></ul><ul><ul><li>base pair deletions, </li></ul></ul><ul><ul><li>mutations, </li></ul></ul><ul><ul><li>inversions, </li></ul></ul><ul><ul><li>translocations and transpositions </li></ul></ul><ul><li>which result in the loss or gain of a recognition site resulting in a fragment of different length and polymorphism. </li></ul>
    15. 15. RFLP <ul><li>6-cutter GAATTC 4-cutter TCGA </li></ul><ul><li>CTTAAG AGCT </li></ul>Enzymes cut DNA at specific sequences Restriction sites are often palindromes:
    16. 18. <ul><li>Advantages: </li></ul><ul><li>variants are co-dominant; </li></ul><ul><li>measures variation at the level of DNA sequence, not protein sequence. </li></ul><ul><li>Disadvantages: </li></ul><ul><li>labor intensive; </li></ul><ul><li>requires relatively large amounts of DNA </li></ul>Using RFLP polymorphism
    17. 19. PCR Based Molecular Markers <ul><li>2. Randomly amplified polymorphic DNA Markers (RAPD) </li></ul><ul><li>RAPD was the first PCR based molecular marker technique developed and it is by far the simplest. </li></ul><ul><li>Short PCR primers (approximately 10 bases) are randomly and arbitrarily selected to amplify random DNA segments throughout the genome. </li></ul>
    18. 20. PCR Based Molecular Markers <ul><li>The resulting amplification product is generated at the region flanking a part of the 10 bp priming sites in the appropriate orientation. </li></ul><ul><li>RAPD often shows a dominant relationship due to primer being unable to bind on recessive alleles. </li></ul><ul><li>RAPD products are usually visualized on agarose gels stained with ethidium bromide. </li></ul>
    19. 21. RAPD: Randomly amplified polymorphic DNA Size sorted
    20. 22. RAPDs <ul><li>Advantages: </li></ul><ul><li>fast, </li></ul><ul><li>relatively inexpensive, </li></ul><ul><li>highly variable. </li></ul><ul><li>Disadvantages: </li></ul><ul><li>markers are dominant. </li></ul><ul><li>Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which. </li></ul><ul><li>Data analysis more complicated. </li></ul>
    21. 23. RAPD Analysis B
    22. 24. 3. Simple Sequence Repeats (SSR)/Microsatellites <ul><li>Simple sequence repeats are present in the genomes of all eukaryotes and consists of several to over a hundred repeats of a 1-4 nucleotide motif. </li></ul>
    23. 26. 4. Amplified Fragment Length Polymorphism (AFLP) <ul><li>AFLP is the latest form of marker assisted selection and is a highly sensitive method based on the combined concepts of RFLP and RAPD </li></ul><ul><li>This technique is applicable to all species giving very reproducible results </li></ul><ul><li>The basis of AFLP is the PCR amplification of restriction enzyme fragments of genomic DNA </li></ul>
    24. 28. AFLPs
    25. 29. AFLPs <ul><li>Advantages: </li></ul><ul><li>fast, </li></ul><ul><li>relatively inexpensive, </li></ul><ul><li>highly variable. </li></ul><ul><li>Disadvantages : </li></ul><ul><li>markers are dominant. </li></ul><ul><li>Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which . </li></ul>
    26. 30. VNTR: variable number tandem repeats <ul><li>Non-coding regions </li></ul><ul><li>Several to many copies of the same sequence </li></ul><ul><li>Large amount of variation among individuals in the number of copies </li></ul>
    27. 31. VNTR Mini satellite
    28. 33. Microsatellites <ul><li>Design primers to flanking regions </li></ul>
    29. 34. Microsatellites <ul><li>Used for within-population studies; </li></ul><ul><li>not as much for between-population studies b/c they evolve too fast </li></ul><ul><li>Paternity analysis and other studies of kinship </li></ul>
    30. 35. Microsatellites <ul><li>Advantages: </li></ul><ul><li>highly variable, </li></ul><ul><li>fast evolving, </li></ul><ul><li>Disadvantages: </li></ul><ul><li>Relatively expensive and time consuming to develop </li></ul>
    31. 36. What is a SNP?
    32. 37. SNP Key Concepts <ul><li>Definition: More than one alternative bases occur at an appreciable frequency </li></ul><ul><li>Availability: Over 10 million SNPs have been identified in human genome (dbSNP Build 125) </li></ul><ul><li>Function: Most SNPs are neutral, and less than 1% is present in protein-coding regions </li></ul>
    33. 38. SNP <ul><li>The most common genetic polymorphism </li></ul><ul><li>Distribute throughout genome with high density </li></ul><ul><li>More stable and easy to assay </li></ul><ul><li>Major cause of genetic diversity among different (normal) individuals, e.g. drug response, disease susceptibility. </li></ul><ul><li>Facilitates large scale genetic association studies as genetic markers . </li></ul>
    34. 39. SNP Types
    35. 41. Application of MAS <ul><li>Parentage determination </li></ul><ul><li>Estimation of genetic distance </li></ul><ul><li>Sex determination </li></ul><ul><li>Identification of carrier stage </li></ul><ul><li>Gene mapping </li></ul>
    36. 42. Polymerase Chain Reaction
    37. 43. Polymerase chain reaction <ul><li>Polymerase chain reaction was invented by Kary Mullis and his colleagues in the 1983. Nobel prize 1993 </li></ul><ul><li>It has become the most widely used nucleic acid amplification technology </li></ul><ul><li>PCR is a technique for in vitro amplification of specific DNA sequences </li></ul>
    38. 44. . <ul><li>Temperature mediated DNA polymerase enzyme by simultaneous primer extension of complementary strands of DNA </li></ul><ul><li>PCR is a test tube system for DNA replication that allows a &quot;target&quot; DNA sequence to be selectively amplified, several million-fold in just a few hours </li></ul>
    39. 45. Standard Polymerase Chain Reaction
    41. 47. PCR Reaction Components <ul><li>Template: previously isolated and purified. </li></ul><ul><li>Two primers: to flank the target sequence. </li></ul><ul><li>Four deoxynucleosides triphosphate (dNTPs): to provide energy and nucleosides for the synthesis of DNA. </li></ul><ul><li>Buffer system containing magnesium. </li></ul><ul><li>DNA polymerase </li></ul>
    42. 48. PCR Reaction Components <ul><li>Template The recommended amount of template for standard PCR is : </li></ul><ul><li>The maximum amount Animal genomic DNA should be up to 500 ng. </li></ul><ul><li>1-10 ng bacterial DNA. </li></ul><ul><li>0.1-1 ng plasmid DNA. </li></ul><ul><li>Primers: Primer concentration between 0.1 and 0.6  M are generally optimal. </li></ul><ul><li>Higher primer concentrations may promote mispriming and accumulation of non specific product </li></ul><ul><li>Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yield of desired product </li></ul><ul><li>Deoxynucleosides Triphosphate (dNTPs) Concentration </li></ul><ul><li>Balanced solution of all four dNTPs minimize polymerase error rate </li></ul>
    43. 49. <ul><li>Buffer system containing magnesium: </li></ul><ul><li>Providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Generally, the Ph of the reaction buffer is (Ph 8.3 – 9.0) will give the optimal results. </li></ul><ul><li>Mg+ Concentration </li></ul><ul><li>The optimal MgCl 2 concentration may vary from approximately 1mM-5mM, 1.5 mM is optimal in most cases. </li></ul><ul><li>DNA Polymerase: </li></ul><ul><li>The recommended amount is 0.5 – 2.5 units/50  l reaction. Too little will limit the amount of product, while too much can produce unwanted non specific products and decreased specificity. </li></ul>
    44. 50. DNA Polymerase <ul><li>The most widely characterized polymerase is that from Thermus aquaticus (Taq DNA Polymerase), which is a thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C  . </li></ul><ul><li>The purified protein (Taq enzyme) consist of a single polypeptide chain has a molecular weight of 95 Kd, and has an optimum polymerization temperature of 70 – 80 C  . </li></ul>
    45. 51. <ul><li>Initial Denaturation: </li></ul><ul><li>This step consists of heating the reaction to a temperature of 94-95°C which is held for 1-9 minutes </li></ul><ul><li>It is enough to completely denature complex genomic DNA </li></ul><ul><li>Each cycle includes three successive steps: </li></ul><ul><li>Each cycle takes as little as few minutes and it usually takes fewer than 20 cycles to produce as much amplified DNA as one needs </li></ul><ul><li>Denaturation: One to several minutes at 94-96 C  , during which the DNA is denatured into single strands </li></ul><ul><li>Annealing: One to several minutes at 50-65 C  , during which the primers hybridize or &quot;anneal&quot; (by way of hydrogen bonds) to their complementary sequences on either side of the target sequence </li></ul><ul><li>Extention: For fragments up to 3 kb, primer extension is normally carried out at 72 C  </li></ul><ul><li>Polymerase binds and extends a complementary DNA strand from each primer and add approximately 60 bases per second at 72C  . </li></ul>Thermal Cycling Profile for Standard PCR
    46. 52. Denaturation Annealing Extention
    47. 53. Number of Cycles: <ul><li>The number of cycles required for optimum amplification varies depending on the amount of the starting material. </li></ul><ul><li>In optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide. </li></ul><ul><li>Most PCR should , therefore, include only 25 – 35 cycles. </li></ul><ul><li>As cycle increases, nonspecific products can accumulate. </li></ul><ul><li>After 20- 40 cycles of heating and cooling build up over a million copies of original DNA molecules. </li></ul>
    48. 55. PCR Phases <ul><li>Three phases: </li></ul><ul><li>Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise </li></ul><ul><li>Linear: The reaction components are being consumed, the reaction is slowing, and products are starting to degrade </li></ul><ul><li>Plateau: The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade </li></ul>
    49. 56. <ul><li>Advantages of PCR: </li></ul><ul><li>Useful non- invasive procedure. </li></ul><ul><li>Simplicity of the procedure. </li></ul><ul><li>Sensitivity of the PCR. </li></ul><ul><li>Disadvantages of PCR: </li></ul><ul><li>False positive results (cross contamination) </li></ul><ul><li>False negative results (e.g. rare of circulating fetal cells) </li></ul>
    50. 57. Variants of PCR <ul><li>Reverse transcriptase-PCR </li></ul><ul><li>Nested-PCR. </li></ul><ul><li>Hot-start PCR </li></ul><ul><li>Quantitative PCR </li></ul><ul><li>Multiplex-PCR </li></ul><ul><li>Mutagenesis by PCR </li></ul><ul><li>Allele specific PCR </li></ul><ul><li>Inverse PCR </li></ul><ul><li>Asymmetric PCR </li></ul><ul><li>In Situ PCR </li></ul>
    51. 58. Reverse Transcriptase-PCR <ul><li>RT-PCR, one of the most sensitive methods for the detection and analysis of rare mRNA transcripts or other RNA present in low abundance. </li></ul><ul><li>RNA cannot serve as a template for PCR, so it must be first transcribed into cDNA with reverse transcriptase and the cDNA copy is then amplified. </li></ul><ul><li>The technique is usually initiated by mixing short (12-18 base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA . </li></ul>
    52. 60. Nested PCR <ul><li>Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment. </li></ul><ul><li>The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. </li></ul><ul><li>Nested PCR is a very specific PCR amplification . </li></ul>
    53. 61. Hot Start PCR <ul><li>Hot Start PCR significantly improve specificity, sensitivity and yield of PCR </li></ul><ul><li>Some components essential for polymerase activity is separated from the reaction mixture until the temperature in the tubes has exceeded the optimal primer annealing temperature usually 55-65 C ˚ </li></ul><ul><li>Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step . </li></ul>
    54. 62. Quantitative PCR <ul><li>The determination or quantitation of the number of copies of a given gene achieves accurate estimation of DNA and RNA targets. </li></ul><ul><li>Real Time PCR </li></ul><ul><li>Traditional PCR has advanced from detection at the end-point of the reaction. to detection while the reaction is occurring Real-Time PCR is used. </li></ul><ul><li>Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated . This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles. </li></ul>
    55. 63. <ul><li>The real time system reduces the time required for PCR amplification and analysis from hours to minutes. </li></ul><ul><li>Monitor amplification online and in real-time. </li></ul><ul><li>Quickly and accurately quantify results. </li></ul><ul><li>Analyze melting characteristics of PCR product. </li></ul><ul><li>The recent development of real time PCR clearly demonstrates many advantages over other existing method with: high accuracy, wide dynamic range , specificity , sensitivity , reduced carry over contamination and rapid accurate and simultaneous quantification of multiple samples. </li></ul>
    56. 64. <ul><li>Multiplex PCR , made it possible to compare two or more complex genomes, for instance to detect chromosomal imbalances. </li></ul><ul><li>Combining in situ hybridization with PCR made possible the localization of single nucleic acid sequences on one chromosome within an eukaryotic organism. </li></ul><ul><li>Amplification of archival and forensic material. </li></ul><ul><li>Identify testing for transplantation, HLA Typing. </li></ul><ul><li>PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology. </li></ul>Multiplex PCR
    57. 65. Polymerase Chain Reaction <ul><li>The polymerase chain reaction (PCR) is a technique widely used in : </li></ul><ul><li>Molecular biology, </li></ul><ul><li>Microbiology, </li></ul><ul><li>Genetics, </li></ul><ul><li>Diagnostics clinical laboratories, </li></ul><ul><li>Forensic science, </li></ul><ul><li>Environmental science, </li></ul><ul><li>Hereditary studies, </li></ul><ul><li>Paternity testing, and </li></ul><ul><li>Many other applications……………………………………… </li></ul>
    58. 66. <ul><li>DNA SEQUENCING </li></ul>
    59. 67. How DNA Sequence Is Determined? Polyacrylamide Gel Electrophoresis T A G C T A C G DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis But these bands can’t tell us the identity of the terminal nucleotides If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence Juang RH (2004) BCbasics ATC 32 P AT 32 P A 32 P ATCG 32 P ATCGA 32 P ATCGAT 32 P ATCGATC 32 P ATCGATCG 32 P ATCGATCGA 32 P ATCGATCGAT 32 P
    60. 68. How to Obtain DNA Fragments AT ATCGAT Specific Reaction to G A Terminated Biosynthetic method Chemical method Template or Non-radioactive (invisible) Producing various fragments
    61. 69. Phosphodiester bond 3’ 5’ di deoxy nuceotide Terminated dd NTP Sanger’s Method: How Terminated Normal Linking Can not react Juang RH (2004) BCbasics P R P R P R P R P R P R OH 5’ 3’ 1 A 1 2 3 4 5 6 A PO 4 2- H 3’ 5’ 2 H
    62. 70. Application of DNA sequencing technology <ul><li>Determination of exact order of bases i.e. deciphering the genetic blueprint of humans, plants, animals and micro-organisms. </li></ul><ul><li>Conservation of gene sequences </li></ul>
    63. 71. GENE CLONING
    64. 72. Gene Transferred by Plasmid Plasmid gets out and into the host cell Resistant Strain New Resistance Strain Non-resistant Strain Plasmid Enzyme Hydrolyzing Antibiotics Juang RH (2004) BCbasics Drug Resistant Gene mRNA
    65. 73. Target Genes Carried by Plasmid 1 plasmid 1 cell Recombinant Plasmid Transformation Target Gene Recombination Restriction Enzyme Restriction Enzyme Chromosomal DNA Target Genes DNA Recombination Transformation Host Cells
    66. 74. Amplification and Screening of Target Gene 1 1 cell line, 1 colony X100 X1,000 Plasmid Duplication Bacteria Duplication Plating Pick the colony containing target gene =100,000
    67. 76. Species cloned <ul><li>Cattle : Alpha and Beta (males, 2001) and (2005) Brazil </li></ul><ul><li>Cat : CopyCat &quot;CC&quot; (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons </li></ul><ul><li>Mule : Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone. </li></ul><ul><li>Horse : Prometea, a Haflinger female born 28 May </li></ul><ul><li>Water Buffalo : Samrupa was the first cloned water buffalo. It was born on February 6, 2009, at India's Karnal, NDRI but died five days later due to lung infection. </li></ul><ul><li>Camel : (2009) Injaz, is the first cloned camel2003, was the first horse clone. </li></ul>
    68. 77. Application of gene cloning <ul><li>Identifying, localizing and characterizing genes </li></ul><ul><li>Creating animal models for studying </li></ul><ul><ul><li>genetic diseases </li></ul></ul><ul><ul><li>Aging </li></ul></ul><ul><ul><li>Cancer </li></ul></ul><ul><ul><li>therapeutic effects of drugs </li></ul></ul><ul><li>Providing animal researchers a tool for saving endangered species </li></ul>
    69. 78. Transgenesis <ul><ul><li>Process of introducing foreign or exogenous </li></ul></ul><ul><ul><li>DNA into an animal’s genome </li></ul></ul><ul><ul><li>Transgene : DNA introduced </li></ul></ul>
    70. 79. <ul><li>Mice </li></ul><ul><li>Cows </li></ul><ul><li>Fish </li></ul><ul><li>Birds </li></ul><ul><li>Sheep </li></ul><ul><li>Goats </li></ul>
    71. 80. Why Transgenesis? <ul><li>Improve genetic Features of domesticated Animals </li></ul><ul><ul><li>Provide animal models for study of human diseases </li></ul></ul><ul><ul><ul><li>Pharming </li></ul></ul></ul><ul><li>using farm animals for production of human pharmaceuticals </li></ul><ul><li>-mammary glands </li></ul><ul><ul><ul><ul><li>Study the genes regulation, development of animals </li></ul></ul></ul></ul>How to Get the Transgene Inserted <ul><li>Retroviral Vectors </li></ul><ul><li>Microinjection </li></ul><ul><li>Embryonic stem cells </li></ul>
    72. 81. Microinjection <ul><li>Remove eggs </li></ul><ul><li>B. Fertilize in vitro </li></ul><ul><li>C. DNA is microinjected into male pronucleus (prior to nuclear fusion) </li></ul><ul><li>100-1000 copies of gene </li></ul><ul><li>D. Implant eggs into surrogate </li></ul>
    73. 82. Fig 22.13 The production of transgenic animals by microinjection of DNA into fertilized eggs. © 2003 John Wiley and Sons Publishers
    74. 83. Retroviral Vectors Infect early stage embryo with replication-defective retrovirus <ul><li>Limitations </li></ul><ul><li>only small DNA inserts </li></ul><ul><li>no regulatory sequences </li></ul><ul><li>safety </li></ul>
    75. 84. Engineered Embryonic Stem Cells <ul><li>Remove pluripotent ES cells from blastocyst </li></ul><ul><li>Transfect ES </li></ul><ul><li>Selection </li></ul><ul><li>Microinject back into blastocyst </li></ul><ul><li>Implant </li></ul>
    76. 85. Mice make Human Antibodies <ul><li>YACs contained many of these heavy and light chain segments </li></ul><ul><li>Knock out Mouse Segments, replace with Human segment genes </li></ul><ul><li>Fully human antibody made </li></ul>
    77. 86. Transgenic Cattle <ul><li>Applications </li></ul><ul><ul><li>Increasing casein content of milk increase cheese production </li></ul></ul><ul><ul><li>Lactose free milk (transgene lactase) </li></ul></ul><ul><ul><li>Resistance to bacterial infections </li></ul></ul><ul><ul><li>In vivo immunization </li></ul></ul><ul><ul><ul><li>transgene is specific Heavy and Light chain genes which </li></ul></ul></ul><ul><ul><ul><li>create An production against a specific antigen </li></ul></ul></ul>
    78. 87. Why Express rProtein in Milk Easy to purify - few other proteins in milk Doesn’t harm transgenic animal- no change to physiology rProtein is authentically modified post-translationally Large quantities Renewable source
    79. 88. Sheep and Pigs PIGS PST porcine somatotropin (growth hormone) adverse effects- kidney, stomach, heart, sterility human Hemoglobin to replace whole blood transfusions SHEEP Increase wool production keratin promoter growth factor
    80. 89. Organ Transplant Pr Problem: Rejection Antibodies from Host bind to Donor Organ Elicits Inflammatory Response Transplanted Organ Lost Solution: Transgene in Donor for Complement-Inhibiting Protein 111
    81. 90. Birds and Fish Birds traditional methods can not be used because of avian embryogenesis differences no ES cells found ALV resistant chickens transgene - defective ALV genome makes viral RNA and protein but blocks assembly of viral particles Fish aquaculture transgene - growth hormone
    82. 91. Application of Transgenesis <ul><li>Production of bio-molecules in an animal which it normally does not produces </li></ul><ul><li>Promoting productivity </li></ul><ul><li>Reducing costs (disease resistance) </li></ul>
    83. 94. Molecular genetics - POULTRY <ul><li>Chickens have 78 chromosomes </li></ul><ul><li>The chicken genome comprises 39 pairs of chromosomes </li></ul><ul><ul><li>8 pairs of macro chromosomes </li></ul></ul><ul><ul><li>1 pair of sex chromosome </li></ul></ul><ul><ul><li>30 pairs of micro chromosome </li></ul></ul>
    84. 95. Molecular genetics - POULTRY <ul><li>The size of the chicken genome is estimated to be 1.2× 10 9 base pairs which is App. 4000 cM in length( 1cM =300kb of DNA in chicken) </li></ul><ul><li>Chicken have played a more significant role in molecular genetics than in most areas of biology </li></ul><ul><li>Mendel discovered the working of single genes and chicken were the species in which this basic concept was applied, e.g. in the selection methods for plumage colour. </li></ul>
    85. 96. Genes of interest <ul><li>With the possibility of producing genetically manipulated transgenic chickens, it has become important to identify the genes controlling economically important traits: </li></ul><ul><ul><ul><li>MUSCLE GROWTH </li></ul></ul></ul><ul><ul><ul><li>PRODUCTION </li></ul></ul></ul><ul><ul><ul><li>REPRODUCTION </li></ul></ul></ul><ul><ul><ul><li>DISEASE RESISTANT </li></ul></ul></ul>
    86. 97. Gene structure OVALBUMIN <ul><li>Major protein of egg white </li></ul><ul><li>1 st to be discovered </li></ul><ul><li>Exons (intervening sequences) were located </li></ul><ul><li>5’ end of introns has G-T dinucleotide </li></ul><ul><li>3’ end of introns has A-G dinucleotide </li></ul><ul><li>polymorphismfor restriction sites within introns was found for both an ECoRI site and Hae Ⅲ site </li></ul><ul><li>All this holds good for other eukaryotic genes also. </li></ul>
    87. 98. Gene structure CONALBUMIN <ul><li>Major iron binding protein. So, acts as antimicrobial agent. </li></ul><ul><li>≈ 10% of the protein </li></ul><ul><li>Purified by polysome immuno-precipitation and column chromatography from chicken oviduct & cDNA synthesized </li></ul><ul><li>cDNA was used to demonstrate that the mRNA in linear and oviduct are identical and coded for by the same gene </li></ul>
    88. 99. <ul><li>Is a protease inhibitor </li></ul><ul><li>≈ 10% of egg white protein </li></ul><ul><li>** transcription initiates at 2 sites, one related to a typical TATA box around -30 base pairs and another related to a AT rich region 85 basepairs upstream, the former being more efficient in vivo. </li></ul>Gene structure OVOMUCOID
    89. 100. <ul><li>≈ 2% of egg white proteins </li></ul><ul><li>1 st enzyme whose tertiary structure was determined </li></ul><ul><li>** there are multiple mRNA for chicken lysosome differing at the 5’ end and extending to either 29, 31, or 53 nucleotides from the common initiation codon </li></ul>Gene structure LYSOSOME
    90. 101. The full genome sequence of the chicken <ul><li>2004 -the first draft of the chicken genome assembly </li></ul><ul><li>Large numbers of chicken full-length cDNAs are already being   sequenced </li></ul><ul><li>It has been predicted that the chicken has 35,000   genes in total ~ 50% of these have a known homologue in another species </li></ul>
    91. 102. Some Advances <ul><li>Molecular kits are being generated to study metabolicfunctions and immune responses (MIN  et al.  2003 ; NEIMAN  et   al.  2003 ) </li></ul><ul><li>Analyze global gene expression in target tissues   of chickens (COGBURN  et al.  2003 ) </li></ul><ul><li>There are also projects to target gene function by disrupting and gaining functions   with the use of RNAi methods  (HUDSON  et al.  2002 ; PEKARIK   et al.  2003 ) </li></ul><ul><li>Single nucleotide polymorphisms within chicken genes are being   exploited for the generation of candidate genes for quantitative   traits (EMARA and KIM 2003 ). </li></ul>
    92. 103. Some Advances <ul><li>There is consequently   extensive research into >200 chicken quantitative trait loci   encoding for disease susceptibility, immunology, leanness, egg   production, etc. ( LIU  et al.  2001 ; MARIANI  et al.  2001) </li></ul><ul><li>Chicken DT40 cell lines are avian-leukosis-virus-induced B cell   lines that exhibit a high ratio of targeted to random integration   of transfected DNA constructs at homologous loci (DHAR  et al.   2001 ).  </li></ul><ul><li>A feature of DT40   cell lines, however, is that they have a high degree of chromosomal   rearrangements that, to date, could not be karyotyped. (WINDING and BERCHTOLD 2001 ) </li></ul>
    93. 104. Some Advances <ul><li>Some 15,000 genes are expressed in chicken tissues involved in the regulation and development of immune responses ( ) </li></ul><ul><li>The chicken&quot;s major histocompatibility complex (MHC) haplotype has a profound influence on the resistance or susceptibility to certain pathogens ( Research Project:  USING THE GENOME TO UNDERSTAND IMMUNOGENETICS OF POULTRY ) </li></ul>
    94. 105. Every body fishing for New Ideas
    95. 106. How much we differ from Ape DNA answers ?
    96. 107. April 25th celebrates the DNA Model DNA continues to the Temple of Science
    97. 108. Madam, are you fiddling with GENES ?
    98. 109. Be A GENE Genius Become Famous
    99. 110. REFERENCE <ul><li> </li></ul><ul><li>Bumstead, N. and Palyga, J. (1992) A preliminary linkage map of the chicken genome. Genomics 13: 690-697 . </li></ul><ul><li>Groenen, M.A.M., Cheng, H.H., Bumstead, N., Benkel, B.F., Briles, W.E., Burke, T. et al. (2000).A consensus linkage map of the chicken genome. Genome Research 10: 137-147. </li></ul><ul><li>Molecular genetics in a modern poultry breeding organization J.E. FULTON a </li></ul><ul><li>BURT, D. W., C. BRULEY, I. C. DUNN, C. T. JONES, and A. RAMAGE  et al. , 1999  The dynamics of chromosome evolution in birds and mammals. Nature  402 :411-413. </li></ul><ul><li>EMARA, M. G. and H. KIM, 2003  Genetic markers and their application in poultry breeding. Poult. Sci.  82 :952-957 </li></ul><ul><li>MASABANDA, J., R. FRIEDL, A. SAZANOV, J. M. LAHTI, and H. LI  et al. , 1998  Mapping of five members of the cyclin gene family on chicken chromosomes by FISH. Chromosome Res.  6 :231-233. </li></ul>
    100. 111. REFERENCE <ul><li>SCHMID, M., I. NANDA, M. GUTTENBACH, C. STEINLEIN, and M. HOEHN  et al. , 2000  First report on chicken genes and chromosomes 2000. Cytogenet. Cell Genet.  90 :169-218 </li></ul><ul><li>TATSUDA, K. and K. FUJINAKA, 2001  Genetic mapping of the QTL affecting body weight in chickens using a F2 family. Br. Poult. Sci.  42 :333-337. </li></ul><ul><li>WINDING, P. and M. W. BERCHTOLD, 2001  The chicken B cell line DT40: a novel tool for gene disruption experiments. J. Immunol. Methods  249 :1-16 </li></ul>