transgenic animals 2

8/4/2019 Transgenic Animals 2

1/15

Using DNA Technology to Produce

Transgenic Animals


2/15

Transgenic Animals


3/15

Transgenic Animals: A Focuson Transgenic Mice Studies


4/15

I. IntroductionTransgenic animals:

Animals which have been genetically engineered tocontain one or more genes from an exogenous source.

Transgenes are integrated into the genome.

Transgenes can be transmitted through the germline toprogeny.

First transgenic animal produced = Founder Animal


5/15

II. Introduction of foreign genes

into intact organisms

Procedure is basically the same regardless ofwhich animal is involved.

Integration usually occurs prior to DNAreplication in the fertilized oocyte.

Majority of transgenic animals carry the gene in all oftheir cells, including the germ cells. Transmissionto next generation requires germline integration.

Some integration events occur subsequent to DNAreplication giving rise to mosaic animals which may or

may not contain the transgene in its germline.


6/15

III. Procedure for Producing

Transgenic Mice

Three different breeding pairs of mice are

required.


7/15

First Breeding Pair:

Fertile male + superovulated female

Fertile male = stud (changed regularly to ensure performance)

Superovulated female = immature female induced tosuperovulate

Pregnant mares serum (=FSH) on day 1

Human Chorionic Gonadotropin (=LH) on day 3

Mated on day 3

Fertilized oocytes microinjected on day 4 with foreign DNAconstruct.

Microinjected oocytes are transferred to the oviducts ofsurrogate mothers at end of day 4.


8/15

Second breeding pair:

Sterile male + surrogate mother Sterile male produced through vasectomy

Surrogate mother must mate to be suitable recipient of

injected eggs Mated on day 3

Microinjected oocytes from first breeding pair aretransferred to oviducts on day 4

Embryos implant in uterine wall and are born 19 days later.

Southern blotting techniques confirm presence and copynumber of transgenes.


9/15

Third breeding pair:

Foster parents

Fertile male + female mated to give birth on sameday surrogate mother

Serves as foster parent if caesarian section is

required for surrogate mother


10/15

IV. Manipulation of Fertilized

Oocytes


11/15

V. Gene Expression in

Transgenic Mice

In order to discriminate the products of theinjected gene from those of an endogenouscounterpart, the injected gene must be marked in

some way. Mini-genes where exons are deleted of cDNA where

introns are absent.

Modification by insertion/deletion/mutagenesis of a few

nucleotides (e.g. the gain or loss of a restrictionendonuclease site).

Hybrid genes where foreign epitopes are expressed ontransgenic products.


12/15

VI. Tissue-Specific Gene

Expression

Generally, if a tissue-specific gene is expressed at all, thenit is expressed appropriately, despite the fact that it hasintegrated at a different chromosomal location.


13/15

VII. Examples of Studies

Utilizing Transgenic Mice

Pharm animals (transgenic livestock)

Bioreactors whose cells have been engineered tosynthesize marketable proteins

DNA constructs contain desired gene and appropriateregulatory sequences (tissue-specific promoters)

More economical than producing desired proteins incell culture


14/15

Naked human Hb frompigs

Human lactoferrin in

cows milk Alpha-1-antitrypsin in

sheep

HGH in mouse urine(uroplakin promoters)

Human antibodies in mice(H and L chain tgenicshybridomas)

CfTCR in goats

Tissue plasminogen

activator (TPA) in goats

Human antithrombin III in

goats

Malaria antigens in goats

(vaccine)

Alpha-glucosidase in

rabbits (Pompes disease


15/15

VIII. Transgenic Pigs for the

Production of Organs for

TransplantationPig organs are rejected acutely due to the presence

of human antibodies to pig antigens.

Once human antibodies are bound to pig organs,human complement is activated and triggers thecomplement cascade and organ destruction.

Transgenic pigs with complement inhibitors havebeen produced and are gaining feasibility as asource of xenogeneic organs for transplantation.

transgenic animals 2

Documents