sperm mediated transfer 2012

Eliane's thesis is available on line.

Manipulation of Sperm for Efficient Production of Transgenic Calves and Chicks.

Eliane Harel- Markowitz

http://shemer.mslib.huji.ac.il/dissertations/H/AGR/001520453.pdf

Abstract

The current method of micromanipulation used in domestic animals results in less than 1% transgenic animals, which makes it extremely difficult to produce transgenic calves and is completely unfeasible for producing transgenic chicks. The purpose of this work was to find a more efficient method for producing transgenic calves and chicks using a combination of two techniques, lipofection and restriction enzyme mediated insertion (REMI). Previously investigators were unable to produce transgenic chicks using lipofection alone, while injection of isolated sperm nuclei, which had been incubated with restriction enzyme, into oocytes had only been demonstrated in frogs. In this study, we demonstrated for the first time, that lipofection of both DNA and restriction enzyme could be used to successfully integrate DNA into sperm genome DNA and then used for routine AI to produce transgenic calves and chicks. First it was demonstrated using needle pricking and southern blot analysis of genomic DNA that the restriction enzyme opens up “hot”  spots in the sperm genomic DNA, producing sticky ends into which foreign DNA  could be inserted and integrated into the sperm genomic DNA rather than just free DNA floating in the fertilized egg. The “transgenic sperm” thus made were used in IVF and AI to produce embryos expressing a foreign DNA, GFP (green fluorescent protein). Using Not I and linearized pGFP lipofected to sperm for AI resulted with two calves which expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the GFP protein; (c) homology analysis between GFP DNA and PCR product DNA sequences and (d) Southern blot analysis.  Similarly in the chicken, linearized plasmid GFP sequences with the corresponding restriction enzyme (REMI) were lipofected into the sperm. The transfected sperm were then used for AI in hens and 90% (17/19) of the resultant chicks expressed the exogenous DNA in their lymphocytes as determined by  (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the GFP and (c)  Southern blot analysis. A complete homology was found between the Jellyfish GFP DNA and a 313 bp PCR product of DNA from chick blood cells. The procedure was then tested with an additional construct, hFSH. The construct of hFSH consisted of both subunits, alpha and beta and the PCR product used primers for both alpha and beta subunit resulted with a PCR product of 584 bp which was unique to transgenic chickens. The procedure was then used to lipofect a construct of hFSH (Human Follicle Stimulating Hormone) into chicken sperm and used for AI. The resultant offspring were transgenic in at least three generations as determined by (1) measurement of hFSH protein in chicken blood using enzyme immunoassay (EIA) and RIA;  (2) RT-PCR and PCR; and (3) copy number.  We conclude that (1) lipofection of both DNA and restriction enzyme into sperm  (bovine and chicken) induces the integration of the DNA into the sperm genomic DNA; (2) lipofected sperm can be used in AI to produce a high percentage of transgenic calves and chicks; (3) The integrated gene is expressed in first, second and third generation; and (4) the method is not limited to specific genes. The technique of lipofection of DNA combined with REMI is therefore an efficient and stable method for producing transgenic domestic animals.  Efficient production of transgenic domestic animals could have major impact on gene therapy, improving livestock breeds and the production of valuable pharmaceuticals, e.g. hFSH, which could be extracted from eggs and milk.

The e-book Sperm-mediated Gene Transfer: Concepts and Controversies. (ed. K. Smith) is  online.

Sperm-Mediated Gene Transfer: Concepts and Controversies
DOI: 10.2174/97816080523701120101
eISBN: 978-1-60805-237-0, 2012
Editor: Kevin R. Smith  Abertay University

Manipulation of Sperm for Efficient Production of Transgenic Calves and Chicks Pp.92-102

Mordechai Shemesh, Laurence Shore, Yehuda Stram, Eliane Harel-Markowitz and Michael Gurevich

Introduction

Sperm-mediated gene transfer (SMGT) represents a novel set of technologies for animal (or in the future, human) genetic modification using the sperm as a vector, as opposed to more traditional established routes such as fertilized eggs or embryonic stem cells.

Studies of sperm-mediated gene transfer (SMGT) indicate that sperm cells possess the ability to be utilized as carriers of exogenous genetic sequences, offering the potential of a novel cost-effective route for germline genetic modification. The fate of transgenes borne by sperm cells has been inconsistent, and analysis of offspring from SMGT experiments has shown a mixed picture in terms of genomic integration of the transgene, suggesting an episomal mode of inheritance. Various distinct steps in transgene uptake by the sperm cell have been described or proposed, including a model based upon endogenous reverse transcriptase activity. Although mature sperm cells are naturally protected against uptake of foreign nucleic acid molecules, certain environmental conditions, for example at key times within the reproductive tract, may reduce this protection, suggesting that SMGT may occasionally take place in nature. If correct, this carries profound implications for evolution and human genetic health. This e-book brings together theoretical and empirical reviews from experts in SMGT, providing comprehensive coverage of the major trends, developments and controversies in this novel field. This e-book is intended as a reference for professional researchers in the field of animal genetic modification (transgenesis) as well as teachers, scientists and physicians interested in medical genetics in general and gene therapy in particular.

http://www.benthamscience.com/ebooks/9781608052370/index.htm

Shemesh, M, Shore L., Stram Y., Harel-Markowitz, E. and Gurevich M. (2012). Manipulation of sperm for efficient production of transgenic calves and chicks. In: Sperm-mediated Gene Transfer: Concepts and Controversies. (ed. K. Smith) pp. 130-145. Bentham E-book

The current method of micromanipulation used for domestic animals results in less than 1% transgenic animals. This makes it extremely difficult to produce transgenic cows and is not feasible for producing transgenic chickens. The purpose of this work was to find a more efficient method for producing transgenic calves and chicks using a combination of two techniques, lipofection and restriction enzyme mediated insertion (REMI). Previously investigators were unable to produce transgenic chickens using lipofection alone. On the other hand, injection of isolated sperm nucleus incubated with restriction enzyme into oocytes has only been shown to be effective in frogs. In this study, we demonstrated for the first time, that lipofection of both DNA and restriction enzyme could be used to successfully integrate DNA into the sperm genome DNA and then used for routine AI to produce transgenic calves and chicks. First it was demonstrated using needle pricking and southern blot analysis of genomic DNA that the restriction enzyme opens up “hot” spots in the sperm genomic DNA. This produces sticky ends by which foreign DNA can be inserted and integrated into the sperm genomic DNA. The “transgenic sperm” thus made were used in IVF and AI to produce embryos expressing a foreign DNA, EGFP (enhanced green fluorescent protein). Using Not I and linearized pEGFP lipofected to sperm for AI resulted with two calves which expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP protein; (c) homology analysis between EGFP DNA and PCR product DNA sequences and (d) Southern blot analysis. Similarly in the chicken, linearized plasmid EGFP sequences with the corresponding restriction enzyme (REMI) were lipofected into the sperm. The transfected sperm were then used for AI in hens and 90% (17/19) of the resultant chicks expressed the exogenous DNA in their lymphocytes as determined by: (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP; and (c) Southern blot analysis. A complete homology was found between the Jellyfish EGFP DNA and a 313 bp PCR product of DNA from chick blood cells. The procedure was then tested with an additional construct, hFSH. The construct of hFSH consisted of both subunits, α and β and the PCR product used primers for both α and β subunit resulted with a PCR product of 584 bp which was unique to transgenic chickens. The procedure was then used to lipofect a construct of hFSH (Human Follicular Stimulating Hormone) into chicken sperm and used for AI. The resultant offspring were transgenic for at least three generations as determined by: (1) measurement of hFSH protein in chicken blood using enzyme immunoassay and RIA; (2) RT-PCR and PCR; and (3) copy number.

We conclude: (1) that lipofection of both DNA and restriction enzyme into sperm (bovine and chicken) induces the integration of the DNA into the sperm genomic DNA; (2) lipofected sperm can be used in AI to produce a high percentage of transgenic calves and chicks; (3) The integrated gene is expressed in the first, second and third generation; and (4) the method is not limited to specific genes. The technique of lipofection of DNA combined with REMI is therefore an efficient and stable method of producing transgenic domestic animals. Efficient production of transgenic domestic animals could have major impact on gene therapy, improving livestock breeds and the production of valuable pharmaceuticals, e.g. hFSH, which could be extracted from eggs and milk.

Real time RT-PCR for hFSH from second generation transgenic chicken tissues using 1:10 serial dilutions of 0.2 µg/tube of extracted RNA.

The sample amplification curves begin rising between the 35^th and 39^th cycle of PCR, while negative control (not shown) remained horizontal. The differences in the cycle no. of the curves represent differences in the gene expression in different tissues