ANS/PLSS 433: Transgenic Animals and Gene Therapy

	The production of transgenic livestock is a relatively new, 
and a rapidly growing area of interest in Animal Biotechnology. 
The technology is being developed to improve production traits, 
improve animal health, and as a means to produce products important 
in medical and pharmacological industries. Gene Therapy of humans 
will use Molecular Biotechnology to combat genetic diseases.

I.	Transgenic Animals

	1.	Transgenic Animal:  

			An animal whose genetic composition has 
			been altered to include selected genes from 
			other animals or species by methods other 
			than those used in traditional breeding.

		A.	1st Transgenic Mice (1981)

		B.	1st Transgenic Livestock (Sheep, 1985)

			a.	Pigs and Cattle have now 
				been successful

	2.	Steps in producing a transgenic animal.

		A.	Superovulation of Donor animal

			a.	Pregnant Mare Serum Gonadotropin 
				(PMSG)
				--Follicle Stimulating Hormone-like
				--Increase number of developing 						
					follicles
			b.	Human Chorionic Gonadotropin 
				(hCG)
				--Luteinizing Hormone-like
				--Causes ovulation

		B.	Fertilization (in vivo or in vitro) 
			and Collection of oocytes or embryos

			a.	Laparoscopy surgery needed 
				for collection
			b.	Slaughterhouse ovaries also often 				
				used in livestock

		C.	Insertion of Recombinant DNA 
			into Embryo

			a.	Various methods

		D.	In Vitro Maturation of embryo

			a.	Cleavage stage to Morula
			b.	New Technology:  PCR
				--Used to determine if genes is 
					incorporated in blastomere's 
					genome at this stage.

		E.	Embryo Transfer to Recipient animals 	

			a. 	Recipients are estrus synchronized 
				with donor during the previous 
				estrous cycle
			b.	Prostaglandin F2-alpha commonly 				
				used--Causes luteolysis
			c.	Surgical and non-surgical means 
				are used 
				in the transfers.

		F.	Gestation and Parturtion

		G.	DNA from offspring analyzed for 				
			presence of transgene.

			a.	DNA isolated from tail, blood, etc.
			b.	Amplified by PCR if needed
			c.	Analyzed by DNA fingerprinting

		H.	Transgenic animals (Founders) are 				
			usually mated with non-transgenics to 	
			propagate the transgenic line.

			a.	Is gene expressed in germ lines?
			b.	Transgene is inherited in 
				a Mendelian Fashion
			c.	Genes also diluted 
				in Mendelian Fashion

		
	3.	Methods of getting Recombinant DNA 
		into the embryo

		A.	Microinjection of recombinant DNA into 	
			pronuclei (male) of 1 cell embryo or zygote, 
			before syngamy occurs.

			a.	Most successful method in farm species

			b.	Recombinant DNA must have promotor 
				and polyadenylation signals

			c.	Tissue specific promotors work best, 
				viral promoters unspecific.
			
			d.	Inducible promotors gaining 
				more attention
		
		B.	Retroviral Vectors

			a.	Infect early cleavage stage embryo

			b.	Can only incorporate DNA < 8 kb

	 		c.	Dangerous

		C.	Embryonic Stem (ES) Cells

			a.	May eventually replace microinjection

			b.	Technique

				i.	ES cells grown in tissue culture
				ii.	Clonal cell lines selected
				iii.	ES cells injected into inner cell 
					mass of blastocyst
				iv.	Chimeric offspring have transgene
	
			c.	Problem

				i.	Few ES cells exist for other mammals
				ii.	Recently pig ES cell line developed 				
					and used successfully to produce
 					transgenic pig
		
		D.	Sperm has also been proposed as a vector
			
			a.	One successful report

			b.	Can't be repeated

	4.	New Method of Producing Transgenics

		A.	Nuclear Transfer/Cloning

			a.	Example: Dolly the Sheep
			b.	Transfect primary cells
			c.	Tranfer nucleus to oocyte

		B.	Gene Targeting

			a.	Infusion of mammary gland with 
				genetically manipulated virus
				--Similar to retroviral method
			b.	Mitotically active alveoli incorporate 				
				gene (s)

		C.	Spermatogonal Transfer

			a.	Transfect Spermatogonia with 					
				recombinant DNA
			b.	Inject into Testis

	5.	Technical problems with producing transgenic livestock

		A.	Very low efficiency--<1%

		B.	Opaque Ova
			a.	Can't see pronuclei
			b.	Centrifugation helps, but causes 					
				trauma to egg.

		C.	Long Generation Time

		D.	Most farm species can only carry two zygotes to term.

		E.	FDA approval????



II. Transgenic Mice
	
	A. Valuable Research Tool--Basic Research
	B. Model for Diseases

III. Applications of Transgenic Livestock
	
	A. Improved Production Traits
		1. Somatotropin/Growth Hormone
			a. Increase feed efficiency
			b. Increase milk production
			c. Better Lean:Fat Ratios
			d. GH-Transgenic Animals have had problems:
				--Arthritis
				--Decreased Fertility
				--Premature Aging
				Inducible Promoters may alleviate 
					problems
		2. Stimulation of muscle development: c-SKI gene
			a. Increases muscle hypertrophy
			b. Decrease body fat
		3. Wool Production:  Improve Cysteine Utilization
			a. Cysteine is rate-limiting amino acid 
				in wool production.
			b. Transgenics have been tested which have 
				been transfected with bacterial 
				enzymes that better fix sulfur into 
				Cysteine.
	B. Improve Animal Health
		1. Incorporate disease resistance genes	
			a. Inducible Promoters used to turn on when 
				needed
		2. Preformed antibodies
			a. Genetics of antibody production known 
		3. Interferon
			a. Anti-viral & anti-cancer agent
 	C. Transgenic Milk
		1. Why Milk?
			a. Milk is readily collected and available 
				in large quantities.
			b. Protein composition is simple and specific
				--Caseins
				--Whey Proteins
			c. Mammary gland can carry out 
				post-translational modifications that 
				microbial systems cannot.
		2.Dairy Pharming:  Production of medically 
			important products in milk.
			a.Blood Factors:
				--Factor IX(Hemophilia)
				--Protein C (Thrombosis)
				--Hemoglobin (Artificial Blood)
				--Tissue Plasiminogen Activator (Heart 								Attacks)
			b. Human Alpha-1-Antitrypsin (Emphysema)
			c. Peptide Hormones and Growth Factors
		3. Manipulation of components already in milk
			a. Simulate human breast milk 
				(Natural Formula)
			b. Remove Lactose (Lactose Intolerance)
			c. Alter casein for cheese production
	D. Human organ transplant
		1. Incorporate human genes into pig organs to use 
			as transplants
			a. Pig organs similar in size to humans
			b. Incorporated human genes would lower 
				rejection.
			c. Top candidates:
				--Heart, Liver, & Lungs

IV. Other Transgenic Animals

	A. Transgenic Birds & Eggs
		1. Eggs have advantages similar to milk
			a. Ovalbumin is specific protein
			a. Eggs already being used to produce 
				antibodies
		2. Microinjection not possible
			a. Polyspermy--pronuclei that fuse cannot be 
				identified
			b. Cytoplasmic injection doesn't incorporate
			c. Fertilized ovum has many layers
				i.	tough membrane
				ii.	albumin
				iii.	two shell membranes
		3. Blastoderm transfection 
			a. Blastoderm cells transfected by 
				lipofection
				--cationic lipid/transgene complex 
					(liposome)
			b.	Recipient Blastoderm cells irradiated
			c.	Donor Blastoderms injected into 
					subgerminal space. 
	B. Transgenic Fish
		1. More efficient than mammalian microinjection
			a. Cytoplasmic injection
			b. Embyo Development happens in external 
				environment
			c. 35-80% microinjection survival
			d. 10-70% production of transgenics
		2. Growth Hormone Transgenics very successful already
	C. Transgenic Amphibians and Reptiles
		1. Frogs have been successful
			a. Used in Research similar to Mice
		2. Reptiles?
			a. No Jurassic Park.........Yet.

V.	Gene Therapy

	A.	Genetic Treatment of (Human) Disease
		1. Make specific somatic cells transgenic
			a. Human germ cell transgenics are not being done
				--Ethics 											--Safety
				--Technical Problems
		2. Single gene disorders targeted
		3. Human Studies
			a. Preclinical
				--Lab studies
				--Many underway
			b. Phase I trials
				--6-10 human subjects
				--12 approved as of 1992
				--Even less have been implemented
			c. Phase II trials
				--Expanded number of subjects
				--No gene therapy studies have gone 
					this far
			d. Phase III trials
				--Final trials
				--Comprehensive
 	B. Ex vivo therapy
		1. Steps:
			a. Collect cells from affected individual
			b. Correct gene defect by gene transfer
			c. Select and grow genetically corrected 
				(Remedial) Cells
			d. Infuse or transplant cells back into 
				patient
		2. Bone Marrow diseases most likely candidates
			a. Bone Marrow contains ES cells
		3. Universal Donor cells being studied
			a.  Need to be immunologically benign
	C. In vivo therapy
		1. Direct Delivery
		2. Tissue Specific Promoter on expressible remedial 
			gene
		3. Viral Infection
	D. Antisense therapy
		1. Uses antisense DNA to block or lower translation 
			of detrimental mRNAs
		2. Strategies
			a. Antisense oligonucleotides
				--DNA:RNA hybrid
			b. Antisense gene
				--RNA:RNA hybrid
	E. Livestock implications
		1. Same techniques could be used in transgenic milk 
			a. Mammary gland is fast growing, dynamic 
				tissue
		2. Disease therapy probably not economical